JP2019505486A - Use of a C-type natriuretic peptide variant to treat osteoarthritis - Google Patents

Abstract

The present disclosure relates to the use of variants of C-type natriuretic peptide (CNP) for treating osteoarthritis, ameliorating one or more symptoms of osteoarthritis, and treating disorders having an osteoarthritic component. .

Description

FIELD OF THE DISCLOSURE The present disclosure relates to mutations in C-type natriuretic peptide (CNP) for treating osteoarthritis, ameliorating one or more symptoms of osteoarthritis, and treating disorders having an osteoarthritic component. Regarding the use of the body.

BACKGROUND OF THE DISCLOSURE Osteoarthritis (OA) is a degenerative disease of the joint. This is the most common joint disease occurring in an estimated 10% of men over the age of 60 and an estimated 18% of women. Indeed, OA is one of the leading causes of pain and disability in adults in the aging process. This is not a single cause, but a complex disease that is the end point of many predisposing factors, including joint trauma, age, and obesity. The most common pathological changes in OA joints are articular cartilage degradation, synovial inflammation, increased subchondral bone depth, and bone growth. Current pharmaceutical treatment of OA targets the inflammatory aspect of the disease and does not target the underlying degeneration or structural changes of the joint. These anti-inflammatory treatments provide only temporary pain relief. In contrast, treatments that would reduce or reverse joint degeneration would have a longer, lasting and beneficial effect on patient pain and motor performance. Thus, there is a need for pharmaceutical agents that can reduce joint degeneration in OA patients and / or repair articular cartilage and joint structure.

  C-type natriuretic peptide (CNP) (for CNP precursor protein NPPC, GenBank accession number NP_077720) is a small and widely expressed (most notably by the central nervous system, reproductive organs, bone, and vascular endothelium) It is a single chain peptide. CNP binds to two distinct receptors: natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C). NPR-B is a member of the membrane-bound guanylyl cyclase multigene family and produces a second messenger cyclic guanosine monophosphate (cGMP) when activated by CNP binding. In contrast, NPR-C does not have any intracellular signaling domain, but instead functions to clear natriuretic peptides from the extracellular space. Thus, when CNP binds to NPR-C, the receptor is internalized and delivers CNP to the lysosome, where it is degraded. This lowers the effective extracellular concentration of CNP.

  CNP is first generated as a 126 amino acid pre-pro polypeptide from the natriuretic peptide precursor C (NPPC) gene. Removal of the signal peptide yields pro-CNP and further cleavage by the endoprotease furin yields a biologically active 53 amino acid peptide (CNP-53). This is secreted and further processed to yield the mature 22 amino acid peptide (CNP-22). CNP-53 and CNP-22 differ in their distribution. CNP-53 is found primarily in tissues, while CNP-22 is found primarily in plasma and cerebrospinal fluid. The main CNP form in cartilage is not currently known. Both CNP-53 and CNP-22 bind to NPR-B with similar kinetics, and both induce cGMP production in a dose-dependent manner. Downstream signaling mediated by cGMP affects a wide variety of biological processes including endochondral bone formation. Thus, increasing or decreasing the level of any component in this pathway can lead to abnormal bone or cartilage growth. For example, knockout of either CNP or NPR-B in a mouse model results in an atrophic phenotype animal with a shorter long bone and spinal column. In addition, mutations in human NPR-B that block CNP signaling result in dwarfism. In contrast, mice engineered to produce high levels of CNP show long long bones and spinal columns.

  The therapeutic potential of CNP is limited by its short half-life. CNP has two mechanisms: 1) through the action of membrane-bound neutral endopeptidase (NEP), which rapidly degrades CNP, and 2) bound NPR-C that directs CNP to the lysosomes where it is degraded. It is wiped out by what These two clearance mechanisms are responsible for the short half-life of CNP (approximately 2.6 minutes). Continuous infusion was essential to increase CNP concentrations beyond the level typically found in human plasma (about 5 pM). This severely limits its therapeutic potential. To overcome this problem, CNP variants with longer half-lives have been developed. Given the potential role in stimulating cartilage growth, CNP variants represent a new class of therapeutics that provide longer lasting relief to OA patients.

SUMMARY OF THE DISCLOSURE The present disclosure relates to the use of CNP variants to treat primary or secondary osteoarthritis, or one or more osteoarthritis-related symptoms. It is herein described that administration of a CNP variant can increase the range of motion of affected joints and delay or reduce osteoarthritis-related damage to cartilage, calcified cartilage, or subchondral bone in treated animals. Disclosed in the document. In various embodiments, the CNP variant is Gly-CNP-37 or Pro-Gly-CNP-37.

  In various aspects, the disclosure provides a method of treating primary osteoarthritis or secondary osteoarthritis, or one or more osteoarthritis-related symptoms, comprising a CNP variant or a composition comprising a CNP variant. A method of treating primary osteoarthritis or secondary osteoarthritis, or said one or more symptoms of disease. Also contemplated is a method of increasing the growth of cartilage, calcified cartilage, or subchondral bone in a subject, wherein the subject comprises a CNP variant or a composition comprising a CNP variant. Administration. The present disclosure also provides a method for delaying, preventing or inhibiting osteoarthritis-related degeneration of cartilage, calcified cartilage, or subchondral bone, comprising a CNP variant or a composition comprising a CNP variant comprising the same. A method comprising administering to a subject in need is contemplated. The present disclosure also provides a method of expanding range of motion or reducing stiffness in a joint affected by osteoarthritis, comprising administering a CNP variant to a subject in need thereof. provide. Furthermore, the present disclosure provides a method of reducing bone growth body growth in a joint affected by osteoarthritis, comprising administering a CNP variant to a subject suffering from osteoarthritis.

In various embodiments, the CNP variant is selected from the group consisting of:

  In various embodiments, increased growth of cartilage, calcified cartilage, or subchondral bone, or delayed degeneration of cartilage, calcified cartilage, or subchondral bone is a subject's knee, shoulder, elbow, finger, hand. Observed in the wrist, crotch, neck, ankle, spine, and / or lower back of the subject. In various embodiments, cartilage damage or growth is analyzed by chondrocyte death / loss, proteoglycan (PG) loss, and collagen loss or fibrillation.

  Also provided is a method of expanding the range of motion of a joint in a subject with osteoarthritis, comprising administering a CNP variant to a subject in need thereof. In various embodiments, the CNP variant has 10%, 20% range of motion as measured by hip flexion, hip extension, hip abduction, hip adduction, knee flexion, or knee extension, Enlarge 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. It is also believed that the administration of CNP variants improves the symptoms of arthritis, including osteoarthritis, as assessed on various patients and pain scales, such as those described in more detail in the detailed description. In various embodiments, the CNP variant is Gly-CNP-37 or Pro-Gly-CNP-37.

  Yet another aspect of the invention is a method of preventing, inhibiting or delaying the growth or formation of bone growth bodies in a mammal comprising administering a CNP variant to a subject in need thereof. About. In various embodiments, the formation or growth of bone growth bodies is associated with osteoarthritis in the subject. The formation or growth of bone growth and the prevention or inhibition of that formation or growth can be determined over time and during physical treatment of CNP variants by physical measurement of the size of the bone growth. In various embodiments, the CNP variant is Gly-CNP-37 or Pro-Gly-CNP-37.

  Another aspect of the present invention is a method of preventing, inhibiting or delaying abnormal growth of subchondral bone or epiphyseal trabecular bone in a mammal, wherein a CNP variant is administered to a subject in need thereof To a method comprising: In one aspect, abnormal growth of subchondral or epiphyseal bone is manifested by bone thickening. In another aspect, abnormal growth of subchondral bone or epiphyseal trabecular bone is associated with osteoarthritis, osteoporosis, or osteosclerosis. In various embodiments, the CNP variant is Gly-CNP-37 or Pro-Gly-CNP-37.

  Yet another aspect of the invention is a method for preventing, inhibiting or delaying synovitis (ie, synovitis) in a mammal, comprising administering a CNP variant to a subject in need thereof. Relates to the method of including. In one aspect, synovitis is manifested at least by the infiltration of mononuclear cells into the affected joint. In another aspect, synovitis is associated with osteoarthritis. In various embodiments, the CNP variant is Gly-CNP-37 or Pro-Gly-CNP-37.

  In one aspect, the CNP variant is administered intraarticularly. CNP variants are also contemplated to be administered by other routes. Exemplary routes of administration include, but are not limited to, subcutaneous, intraarticular, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, intraperitoneal, intramuscular, intradermal, intrathecal, topical, trans Skin or transmucosal administration may be mentioned.

  In another embodiment, the CNP variant is administered in response to joint trauma or injury. In a further embodiment, the CNP variant is administered within one month of joint trauma or injury. In another embodiment, the CNP variant is administered within one week of joint trauma or injury. In other embodiments, the CNP variant is administered after cartilage degeneration has occurred.

  In various aspects, the disclosure provides a method for assessing the effect of a CNP variant on the level of at least one cartilage-related biomarker in a subject suffering from osteoarthritis, wherein the bone is administered with a CNP peptide or CNP variant. A method comprising assaying the level of at least one cartilage-related biomarker in a biological sample from a subject suffering from arthritis is provided. In one aspect, the CNP variant is administered to the subject prior to assaying the level of at least one cartilage-related biomarker.

  In various aspects, contemplated herein is a method of treating osteoarthritis, or one or more osteoarthritis-related symptoms or signs, comprising administering a CNP variant to a subject, The subject is a method that has been identified as having a high level of at least one cartilage-related biomarker. In various embodiments, the at least one cartilage-related biomarker is CNP, cGMP, collagen type II propeptide and fragments thereof, collagen type II and fragments thereof, syndecan-3, annexin VI, proliferating cell nuclear antigen (PCNA), I A propeptide of type procollagen (PINP) and fragments thereof, collagen type I and fragments thereof, and chondroitin sulfate aggrecan.

  In various aspects, the present disclosure provides for the use of a composition comprising a CNP variant. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent. In one aspect, the composition is a lyophilizate prepared from a formulation comprising citrate / citrate buffer or acetate / acetate buffer having a pH of about 4 to about 6.

  Also contemplated are methods of treatment as described herein further comprising administration of a second agent. In various embodiments, the second agent is selected from the group consisting of anti-inflammatory agents, NSAIDs, corticosteroids, and hyaluronic acid.

In various embodiments, the CNP variant used in the method may be attached to a hydrophobic acid or attached to one or more hydrophobic acids. Non-limiting examples of hydrophobic acids include linear or branched, saturated or unsaturated C _{5 to} C ₁₂ carboxylic acids (eg, pentanoic acid, heptanoic acid, etc.) and natural fatty acids. The hydrophobic acid may be attached to the N-terminus, C-terminus, and / or side chain of one or more amino acid residues. In one aspect, the hydrophobic acid is attached to the N-terminus.

  In yet another aspect, the CNP variant used in the method is a chimeric or fusion protein comprising a CNP variant and a cleavable peptide or cleavable protein, or peptide tag. Exemplary cleavable proteins or cleavable peptides include, but are not limited to, histidine (eg, hexa-His) tag; TAF12: human transcription factor TAF12; KSI: ketosteroid isomerase; MBP: maltose binding protein; β-Gal: β -Galactosidase; GST: glutathione-S-transferase; Trx: thioredoxin; CBD: chitin binding domain; BMPM: BMP-2 mutation, SUMO, CAT, TrpE, staphylococcal protein A, streptococcal protein, starch-binding protein, endo Cellulose binding domain of glucanase A, cellulose binding domain of exoglucanase Cex, biotin binding domain, recA, Flag, c-Myc, poly (His), poly (Arg), poly (Asp), poly (Gln), poly (Phe), poly (Cys), green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, biotin, avidin, streptavidin, antibody epitope, and fragments thereof Can be mentioned.

  In various embodiments, the CNP variant may be a monomer or a dimer. In related embodiments, the monomer of the dimeric CNP variant has a N-terminus at the N-terminus, via a linker, or without a linker, via a linker, or without a linker, the N-terminus at the C-terminus, or a linker. The C-terminus can be attached to the C-terminus via or without a linker.

  In any of the aspects disclosed herein, the CNP variant can have substantially the same or better biological activity as wild-type CNP-22. For example, a CNP variant can retain at least 50%, 60%, 70%, 80%, 90%, 95%, or more of the activity of wild-type CNP-22 or stimulate, for example, the production of cGMP It may have greater activity than CNP-22 for interaction with NPR-B. Alternatively or additionally, the CNP variant may retain at least 50%, 60%, 70%, 80%, 90%, 95%, or more of the activity of wild-type CNP-22, or Endochondral bone, including but not limited to chondrocyte proliferation, chondrocyte differentiation, inhibition of mitogen-activated protein (MAP) kinase / MEK (Raf-1) kinase signaling pathway, and promotion of endochondral ossification It may have greater activity than CNP-22 in modulating growth and chondrocyte activity. In any of the embodiments described herein, the CNP variant is at least 40%, 50%, 60%, 70%, 80%, amino acids 6-22 or 1-22 of wild type CNP-22, It may comprise 90%, 95%, or more identical amino acid sequences.

  In various embodiments, the CNP variant optionally binds to promote cartilage targeting, reduce renal clearance, and / or increase resistance to NEP degradation, eg, at the N-terminus and / or C-terminus. Can have elongation. Such a bond or extension is formed from, for example, poly Asp, poly Glu, cartilage targeting peptide, sialoprotein, PEG, carbohydrate, hydrophobic acid, NPPC, or non-CNP (poly) peptide, or combinations thereof; Or it may contain molecules or sequences derived therefrom.

  It is further contemplated that CNP variants may be conjugated to hydrophobic polymeric or non-polymeric moieties such as heptanoic acid, pentanoic acid, or fatty acids. The hydrophobic moiety can be attached to the side chain of an amino acid residue including, but not limited to, lysine, serine, cysteine, or threonine, or can be attached to the N-terminus and / or C-terminus of a CNP variant.

  In various embodiments, CNP variants useful in the present methods have a pI in the range of about 8 to about 10.5, or about 8.5 to about 10.

  In various aspects, the disclosure provides for a pharmaceutical composition comprising a CNP variant, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. Provide use. In various embodiments, the composition is a sterile pharmaceutical composition suitable for parenteral administration. In some embodiments, the composition comprises a substantially pure CNP variant, eg, at least about 90% or 95% pure. In some embodiments, the composition comprises less than about 5%, 4%, 3%, 2%, 1%, or 0.5% contaminants, such as other human proteins, porcine proteins, or (as desired Contains CNP-53 (or other than CNP variants) or fragments thereof. In certain embodiments, the sterile composition is administered to a subject to treat or prevent any CNP-responsive condition or disorder disclosed herein.

  CNP variants useful herein advantageously retain CNP activity and exhibit increased serum half-life. Retention of CNP activity is, for example, at least about 50% of cNPMP-stimulating activity of CNP-22 under the same in vivo biological effect or at the same concentration (eg, greater than 1 μM CNP peptide, or ED80). , 60%, 70%, 80%, 90%, 95%, or at least about 100% retention. In some embodiments, the CNP variant is at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times the serum half-life compared to CNP-22. , 25-fold, 30-fold, 35-fold, or 40-fold increase.

  In related embodiments, the CNP variants described herein have increased NEP resistance and show increased half-life compared to wild-type CNP-22. In one aspect, the half-life of the CNP variant is about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, compared to wild type CNP-22. Increase by 96%, 97%, 98%, 99%, or about 100%.

  In certain embodiments, the CNP variants described herein used in the methods are about 1.5 times, about 2 times, about 2.5 times, compared to wild type CNP-22, About 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times or more, increasing cGMP production in vitro, increasing cGMP production in vivo, cartilage or bone formation or Increase the level of one or more biomarkers associated with growth in vivo, increase resistance to NEP cleavage in vitro, increase plasma or serum half-life in vivo, increase bioavailability in vivo Or increase cartilage growth or regeneration in vivo, or a combination of such increases.

  In various embodiments, the CNP variants described herein are administered at a dose ranging from about 5 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP variant is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, or 2000 nmol Administered at a dose of / kg or other dose deemed appropriate by the treating physician. In other embodiments, the CNP variant is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190. 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 μg / kg, or about 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 2 mg / kg Or other doses deemed appropriate by the treating physician.

  In various embodiments, the CNP variant is administered in a single treatment or in multiple doses. Multiple doses may be given daily throughout the course of treatment or in multiple doses. In various embodiments, the CNP variant is administered in a single dose or multiple doses daily, every other day, every third day, twice per week, three times per week, weekly, every other week, every third week, every month, It is intended to be administered every 6 weeks, every 2 months, every 3 months, or as deemed appropriate by the treating physician.

  In certain embodiments, the administration of the CNP variant is adjusted to allow for a recovery period following the period of prophylactic or therapeutic treatment. For example, a CNP variant may be administered daily or multiple times per week for a period of time, intraarticularly, subcutaneously, intravenously, or by another mode, followed by a period of no treatment, and The cycle is repeated. In some embodiments, the initial period of treatment (eg, administered daily or multiple CNP variants per week) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks. 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In related embodiments, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the CNP variant dosing regimen is 3 days off daily followed by 3 days off; or 1 week, daily or multiple times per week followed by 3 days off or 1 week off; or 2 weeks daily Or multiple times per week followed by 1 week or 2 weeks off; or 3 weeks, daily or multiple times per week followed by 1 week, 2 weeks, or 3 weeks off; or 4 weeks, 5 weeks, 6 weeks 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, daily or multiple times per week, followed by 1 week, 2 weeks, 3 weeks, or 4 weeks off.

  In additional aspects, the disclosure provides a method of treating an osteoarthritis or disorder having osteoarthritis-related symptoms, comprising administering a CNP variant to a subject and in the subject (eg, in a biological sample from the subject). Monitoring the level of at least one cartilage-related biomarker, wherein increasing or decreasing the level of cartilage-related biomarker is indicative of the therapeutic effect of the CNP variant on the subject. In some embodiments, where the biomarker level is increased in relation to the formation or growth of cartilage, calcified cartilage, or subchondral bone, the increased level of the biomarker is a CNP variant of the subject. Shows therapeutic effect. In other embodiments, where the level of biomarker is decreased in relation to the formation or growth of cartilage, calcified cartilage, or subchondral bone, the decreased level of the biomarker is treated with a CNP variant for the subject Effects.

In a further embodiment, the method of treatment further comprises adjusting the amount (or dose) or frequency of administration of the CNP variant:
(I) The amount (or dose) or frequency of administration of the CNP variant is at the level of at least one cartilage-related biomarker, wherein the level of biomarker that increases in relation to cartilage formation or growth is at the target level Or (ii) the amount (or dose) or frequency of administration of the CNP variant is at the level of at least one cartilage-related biomarker and is related to cartilage formation or growth (Iii) the amount (or dose) or frequency of administration of the CNP variant is the level of at least one cartilage-related biomarker, wherein the level of biomarker that is increased exceeds the target level; If the level of biomarker that decreases in relation to cartilage formation or growth exceeds the target level, Or (iv) the amount (or dose) or frequency of administration of the CNP variant is the level of at least one cartilage-related biomarker that decreases with respect to cartilage formation or growth Is reduced below the target level.

  It is intended that the target level of the biomarker refers to a level or range of levels of the biomarker that is associated with a therapeutic effect in the subject and / or a beneficial effect to alleviate or ameliorate the symptoms or symptoms of the disorder. In certain embodiments, the level of biomarker above or below the target level can be detrimental to the subject.

  In other aspects, the present disclosure contemplates methods for assessing the effects of administration of CNP variants on the formation or growth of cartilage, calcified cartilage, or subchondral bone. In one aspect, the method comprises assaying or measuring the level of at least one cartilage-related biomarker in a subject to which the CNP variant has been administered to determine whether the CNP variant is cartilage, calcified cartilage, or subchondral bone. It is realized to evaluate the effect on in vivo formation and in vivo growth. In a related aspect, an increase in the level of at least one cartilage-related biomarker indicates that administration of a CNP variant has a positive effect on the formation or growth of cartilage, calcified cartilage, or subchondral bone; It may indicate a useful treatment for osteoarthritis and other cartilage-related diseases or disorders associated with decreased CNP activity. Exemplary cartilage-related biomarkers include, but are not limited to, CNP (eg, endogenous levels of CNP-22 or CNP-53), cGMP, collagen type II propeptide and fragments thereof, collagen type II and fragments thereof , Syndecan-3, annexin VI, proliferating cell nuclear antigen (PCNA), propeptide of type I procollagen (PINP) and fragments thereof, collagen type I and fragments thereof, and chondroitin sulfate aggrecan.

  In a further aspect, the disclosure provides a method of assessing the effect of a CNP variant on the level of at least one cartilage-related biomarker in a subject, wherein the cartilage is in a biological sample from a subject to which the CNP variant has been administered. A method comprising assaying or measuring the level of a relevant biomarker is contemplated. In some embodiments, the method further comprises administering a CNP variant to the subject prior to assaying or measuring the level of cartilage-related biomarker.

  In some embodiments of methods related to cartilage-related biomarkers (eg, therapeutic methods, diagnostic methods, and assay methods), the CNP variant is Gly-CNP-37, also referred to as Gly-CNP-37, Or any of the CNP peptides and CNP variants described herein, including the CNP variants shown herein. In certain embodiments of such methods, the CNP peptide or CNP variant is not CNP-22 or CNP-53. In a particular embodiment, the CNP variant is Pro-Gly-CNP-37.

  Any of the above-mentioned CNP variants described herein in the preparation of a medicament for the treatment of osteoarthritis and the symptoms described herein or other physiological signs associated with osteoarthritis Use is also contemplated. Syringes, such as single use syringes or prefilled syringes, sterile seal containers, such as vials, bottles, vessels, and / or kits, optionally containing any of the peptides or polypeptides described above, with appropriate instructions for use. Or a package is also contemplated.

  Each feature described herein is an illustrative, non-limiting illustration of any of the aspects of the present disclosure, and thus can be combined with any other feature described herein Is understood to be intended. For example, a feature may be combined with phrases such as “one aspect”, “a specific aspect”, “some aspects”, “a further aspect”, “a specific exemplary aspect”, and / or “another aspect”. Where described, each of these features may be combined with any other feature described herein, and not all possible combinations need be listed. Where examples of values that fall within a range are disclosed, any of these examples are intended as possible endpoints of the range, any numerical value between such endpoints is intended, and upper and lower endpoints Any and all combinations of are envisioned.

FIG. 1 shows that Gly-CNP-37 administration at low dose (35.5 μg) or high dose (110 μg) showed a significant reduction in cartilage degeneration in the tibia. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 2 shows that the depth ratio was improved in the Gly-CNP-37 treatment group. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 3 is a graph showing that the width of cartilage affected by any degeneration was improved in the Gly-CNP-37 treated group. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 4 shows that Gly-CNP-37 treated animals showed greater than 50% improvement in significant tibial cartilage degeneration. FIG. 5 shows that Gly-CNP-37 at 110 μg significantly reduced total joint score (excluding the femur). ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 6 is a graph representing the total joint score for Gly-CNP-37 treated animals when the femur score is included in the calculation. FIG. 7 is a graph showing inner and outer growth plate thicknesses in Gly-CNP-37 treated and untreated control animals. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 8 is a graph representing the data of FIG. 7 in which the outer thickness is subtracted from the inner thickness to determine the difference between the two. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 9 represents the course of the gait score in Gly-CNP-37 treated rats or untreated control rats. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 10 is a graph showing the area under the curve (AUC) of the walking score of FIG. All values are p ≦ 0.05 for ANOVA for the vehicle. FIG. 11 shows the measurement of total cartilage degeneration width for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 12 shows the measurement of significant cartilage degeneration width for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. For t-test against vehicle SC ^† p <0.05. FIG. 13 shows the mean medial tibial proliferator score for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. For t-test against vehicle SC ^† p <0.05. FIG. 14 shows the average growth plate thickness in micrometers for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. ^* P ≦ 0.05 for ANOVA versus vehicle. For t-test against vehicle SC ^† p <0.05. FIG. 15 shows the mean total joint score (minus femur) for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. ^* P ≦ 0.05 for ANOVA versus vehicle. For t-test against vehicle SC ^† p <0.05. FIG. 16 shows the mean synovitis score for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. FIG. 17 shows the mean change in body weight for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. ^* P ≦ 0.05 for ANOVA versus vehicle. FIG. 18 shows the mean gait score for animals treated with Pro-Gly-CNP-37 or Gly-CNP-37. Values are p <0.05 for t-test against vehicle SC. FIG. 19 shows the time course for the mean plasma concentration of Pro-Gly-CNP-37 in animals after administration of Pro-Gly-CNP-37 via the intra-articular or subcutaneous route for each study group. Show. For each line, the number of corresponding test groups is shown directly above the corresponding data line. FIG. 20 shows the mean total tibial cartilage degeneration width for animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late stage osteoarthritis model. (Treatment administration began on day 21 after surgery and data was collected on day 40). FIG. 21 shows the average significant tibial cartilage degeneration width for animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late stage osteoarthritis model. (Treatment administration began on day 21 after surgery and data was collected on day 40). FIG. 22 shows treatment treatment with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late-stage osteoarthritis model (treatment dose 21 days after surgery). The mean tibial cartilage degeneration score for each and total of the three zones is shown. ^* P <0.05 for KW test (Dunn's post) versus each IA or SC vehicle (day 40) control. For ^MW test against IA vehicle (day 40) ^† p <0.05. For ^MW test against SC vehicle (day 40) ^‡ p <0.05. FIG. 23 shows the treatment of animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late-stage osteoarthritis model (21 days after surgery). Shows the mean tibial cartilage degeneration depth ratio for each of the three zones and the mean. ^* P <0.05 for KW test (Dunn's post) versus each IA or SC vehicle (day 40) control. For ^MW test against IA vehicle (day 40) ^† p <0.05. For ^MW test against SC vehicle (day 40) ^‡ p <0.05. FIG. 24 shows the treatment of animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late-stage osteoarthritis model (21 days after surgery). The total joint score with femur for each of the three zones and the mean is shown. For ^MW test against IA vehicle (day 40) ^† p <0.05. For ^MW test against SC vehicle (day 40) ^‡ p <0.05. FIG. 25 is for animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late stage osteoarthritis model (treatment dose 21 days after surgery). The total joint score without femur for each of the three zones and the mean is shown. For ^MW test against IA vehicle (day 40) ^† p <0.05. For ^MW test against SC vehicle (day 40) ^‡ p <0.05. FIG. 26 shows bone proliferator scores for animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late stage osteoarthritis model. (Treatment started 21 days after surgery, data collected on day 40). FIG. 27 shows the treatment of animals treated with Pro-Gly-CNP-37 (intra-articular (“IA”) and subcutaneous (“SC”)) in a late stage osteoarthritis model (21 days after surgery). Shows the change in weight. ^* P <0.05 for KW test (Dunn's post) versus each IA or SC vehicle (day 40) control. For ^MW test against IA vehicle (day 40) ^† p <0.05.

Detailed Description of the Disclosure The present disclosure relates to methods of treating osteoarthritis, one or more symptoms of osteoarthritis, and other disorders having symptoms or components associated with osteoarthritis using CNP variants.

A. Definitions Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

  The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art. This depends in part on how the value was measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5% of a predetermined value or range. 4%, 3%, 2%, 1%, 0.5%, or 0.05% or less. It is understood that the term “about” or “approximately” applies to each of a series of numbers whenever it precedes a first number in the series of two or more numbers.

Definition of standard chemistry terms may, Carey and Sundberg, Advanced Organic Chemistry , 3 rd Edition, Vols. A and B (Plenum Press, New York 1992) may be found in references including. The practice of this disclosure, unless otherwise stated, is within the skill of the art synthetic organic chemistry, mass spectrometry, preparative analysis chromatography, protein chemistry, biochemistry, recombinant DNA technology, and certain conventional methods of pharmacy. May be used. For example, TE Creighton, Proteins:. Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., 4 th Edition, 2004); Sambrook, et al, Molecular Cloning: A Laboratory Manual ^{(2 nd Edition, 1989);} Methods In Enzymology (.. S. Colowick and N. Kaplan eds, Academic Press, Inc); Remington's Pharmaceutical Sciences, 18 th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990) reference.

  All publications, patents, and patent applications cited herein, whether cited above or below, are hereby incorporated by reference in their entirety.

  “Polypeptides” and “proteins” are composed of amino acid residues, their related, naturally occurring structural variants and non-naturally occurring synthetic analogs linked via peptide bonds or peptide bond isosteres. Refers to a polymer. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms “polypeptide” and “protein” are not limited to the minimum length of the product.

  Conventional notation is used herein to describe the polypeptide sequence: the left end of the polypeptide sequence is the amino terminus; the right end of the polypeptide sequence is the carboxyl terminus.

“Conservative substitution” refers to the replacement of an amino acid in a polypeptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In one embodiment, each of the following groups contains natural amino acids that are conservative substitutions for each other:
(1) Alanine (A), Serine (S), Threonine (T);
(2) Aspartic acid (D), glutamic acid (E);
(3) Asparagine (N), glutamine (Q);
(4) Arginine (R), Lysine (K);
(5) Isoleucine (I), leucine (L), methionine (M), valine (V); and (6) phenylalanine (F), tyrosine (Y), tryptophan (W).

In another embodiment, each of the following groups contains natural amino acids that are conservative in substitution with each other:
(1) Glycine (G), alanine (A);
(2) Aspartic acid (D), glutamic acid (E);
(3) Asparagine (N), glutamine (Q);
(4) Arginine (R), Lysine (K);
(5) Isoleucine (I), leucine (L), methionine (M), valine (V), alanine (A);
(6) phenylalanine (F), tyrosine (Y), tryptophan (W); and (7) serine (S), threonine (T), cysteine (C).

In further embodiments, the amino acids may be grouped as described below.
(1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp;
(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(3) Acidity: Asp, Glu;
(4) Basic: His, Lys, Arg;
(5) Residues affecting skeletal orientation: Gly, Pro; and (6) Aromaticity: Trp, Tyr, Phe, His.

  In one aspect, CNP variants described herein that are useful in the methods are generated via recombinant means using a polynucleotide encoding the CNP variant. A CNP variant expressed by such a polynucleotide may be used to propagate the host cell in a medium under conditions suitable for expression of the polynucleotide encoding the CNP variant and to express the expression product from the host cell or medium. Isolating. The actual expression product can vary slightly from the encoded protein product depending on any post-translational processing.

  “Polynucleotide” refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring biopolymers (with respect to molecular types, not necessarily with respect to biopolymer sequences), such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), and nucleic acid analogs. . “CDNA” refers to a single-stranded or double-stranded form of DNA that is complementary to or identical to mRNA.

  “Expression control sequence” refers to a nucleotide sequence that regulates the expression of a nucleotide sequence operably linked thereto. “Functionally linked” is a functional relationship between two parts where the activity of one part (eg, the ability to modulate transcription) results in an action on the other part (eg, transcription of the sequence). Point to. Expression control sequences include, but are not limited to, promoter (eg, inducible or constitutive promoter), 3′UTR, enhancer, transcription terminator, initiation codon (ie, ATG), splicing signal for intron, and stop codon sequence Can be mentioned.

  “Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. Amplified or assembled recombinant polynucleotides can be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell comprising a recombinant polynucleotide is referred to as a “recombinant host cell”. The gene is then expressed in a recombinant host cell, eg, to produce a “recombinant polypeptide”. Recombinant polynucleotides can similarly perform non-coding functions (eg, promoters, origins of replication, ribosome binding sites, etc.).

  As used herein, a “chimera” is a polynucleotide comprising at least two heterologous polynucleotide or polypeptide sequences (ie, not derived from different sources or related to each other as naturally occurring sequences). Or refers to polypeptides, which are attached or linked directly or indirectly using techniques generally known in the art, eg, recombinant expression or chemical cross-linking. In one aspect, the heterologous sequence may comprise a protein or peptide that is directly or indirectly linked to the CNP variant, and includes a protein or peptide that is cleavable from the CNP variant. In related embodiments, the CNP variant is a chimera as described herein.

  In certain embodiments, the chimera includes a CNP fusion protein comprising a cleavable carrier protein or peptide tag. The term “cleavable carrier protein” or “cleavable peptide tag” can be fused directly or indirectly through a linker to a heterologous polypeptide sequence and from a heterologous polypeptide or protein. A peptide or polypeptide sequence that is removable from a heterologous sequence with an agent that cleaves or separates the cleavable peptide or polypeptide. In some embodiments, the cleavable carrier protein or peptide tag improves the production, purification, and / or detection of the fusion protein or heterologous polypeptide. Exemplary cleavable carrier proteins and peptide tags include, but are not limited to, human transcription factor TAF12 (TAF12), ketosteroid isomerase (KSI), maltose binding protein (MBP), β-galactosidase (β-Gal), Glutathione-S-transferase (GST), thioredoxin (Trx), chitin-binding domain (CBD), BMP-2 mutation (BMPM), SUMO, CAT, TrpE, staphylococcal protein A, streptococcal protein, starch-binding protein, Cellulose binding domain of endoglucanase A, cellulose binding domain of exoglucanase Cex, biotin binding domain, recA, FLAG®, c-Myc, poly (His), poly (Arg), poly (A sp), poly (Gln), poly (Phe), poly (Cys), green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, biotin, avidin, streptavidin, antibody epitopes, and fragments thereof It is done.

By “range of motion” is meant the full mobility of the joint, typically the range of flexion and / or extension. The range of motion is typically measured using a goniometer and indicated in arc degrees. Any measurement method known to those skilled in the art can be used to measure the range of motion of the subject's joint. How one illustrative measuring the range of motion of the joint, for example, Norkin, CC and White DJ, Measurement of joint motion: provided a guide to goniometry (FA Davis Company , 2 nd ed Philadelphia.) By (1995) The

  By “joint stiffness” is meant a sense of difficulty moving the joint, or an apparent loss of range of motion of the joint.

  A “cleaving agent” is an agent useful for, for example, cleaving or separating a cleavable peptide or polypeptide from a heterologous polypeptide or protein. Cleavage agents include, but are not limited to, palladium, cyanogen bromide (CNBr), formic acid, hydroxylamine, clostripain, thrombin, chymotrypsin, trypsin, trypsin-like protease, carboxypeptidase, enterokinase (enteropeptidase), Kex2 protease , Omp T protease, factor Xa protease, subtilisin, proTEV, SUMO protease, V8 protease, HIV protease, rhinovirus protease, fliricin protease, IgA protease, human Pace protease, collagenase, Nia protease, poliovirus 2Apro protease, poliovirus 3C Protease, Genenase, Furin, Elastase, Proteinase K, Pepsi , Rennin (chymosin), microbial aspartic protease, papain, calpain, chymopapain, ficin (ficaine), bromelain (bromelase), cathepsin B, caspase, thermolysin, endoprotease Arg-C, endoprotease Glu-C, endoprotease Lys- C, kallikrein, and plasmin.

  The terms “identical” and percent “identity” in the context of two or more polynucleotide or polypeptide sequences are the same or a specified percentage when compared and aligned for maximum correspondence. Refers to two or more sequences or subsequences in which the nucleotide or amino acid residues are the same.

  The phrase “substantially homologous” or “substantially identical” in the context of two nucleic acids or polypeptides generally has at least 40% nucleotide or amino acid residue identity when comprehensively aligned, Refers to two or more sequences or subsequences that are 50%, 60%, 70%, 80%, 90%, 95%, or 98%.

  Comprehensive alignment of sequences for comparison is performed using the EMBOSS Needle program (eg, performed by EMBL-EBI via a web interface) using default parameters (for proteins: matrix = BLOSUM62, gap open = 10, gap Stained = 0.5, output format = pair, end gap penalty = false, end gap open = 10, end gap extended = 0.5; for nucleotides, matrix = DNA full, gap open = 10, gap extended = 0.5, output format = pair, end gap penalty = false, end gap open = 10, end gap extended = 0.5).

  A further indication that two nucleic acid sequences or polypeptides are substantially homologous or identical is that the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid, as described below. It is immunologically cross-reactive with the polypeptide. Thus, a polypeptide is typically substantially identical to a second polypeptide, eg, if the two polypeptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described herein.

  “Substantially pure” or “isolated” is the main species present (ie, richer on a molar basis than any other individual macromolecular species in the composition) And the substantially purified fraction is a composition in which the subject species constitutes at least about 50% (mole basis) of all macromolecular species present. In various embodiments, the species of interest is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the polymeric species present in the composition on a molar or weight basis. Configure. If no contaminant macromolecular species can be detected in the composition by conventional detection methods, the target species is purified to “essential uniformity”. Solvent species, small molecules (<500 Daltons), stabilizers (eg BSA), and elemental ion species are not macromolecular species considered for the purposes of this definition. In one aspect, the compounds of the present disclosure are substantially pure or isolated. In another embodiment, the compounds of the present disclosure are substantially pure or isolated with respect to the polymeric starting material used in their production. In yet another embodiment, a pharmaceutical composition of the present disclosure includes one or more pharmaceutically acceptable excipients, carriers, or diluents, and optionally another biologically active agent. Includes mixed, substantially pure, or isolated CNP variants.

  “Wild type” (wt) is a term that refers to the natural form (including sequence) of a polynucleotide, polypeptide, or protein in a species. Wild-type forms are distinguished from mutant forms of polynucleotides, polypeptides, or proteins that arise from genetic mutations.

  In one embodiment, a first polypeptide that is an “analog” or “variant” or “derivative” of a second polypeptide has at least about 50%, 60% sequence identity with the second polypeptide. Or a polypeptide that is 70% but less than 100% sequence identity. Such analogs, variants, or derivatives may be composed of non-naturally occurring amino acid residues, including but not limited to homoarginine, ornithine, penicillamine, and norvaline, as well as naturally occurring amino acid residues. . Such analogs, variants, or derivatives may be composed of one or more D-amino acid residues and may also be peptidomimetics or peptide-bound isosteres, such as between two or more amino acid residues. Or it may contain non-peptide bonds between peptidomimetic residues. In another embodiment, if the first polypeptide is not a known cleavage product of the second polypeptide or is not a known precursor of the second polypeptide, then the first polypeptide A first polypeptide is an “analog”, “variant”, or “derivative” of a second polypeptide, even if the sequence identity to the two polypeptides is 100% or has a wild-type sequence. It is.

  In one aspect, the term “derived from” as used herein is based on a wild-type or naturally occurring polypeptide sequence or peptide sequence, and one or more deletions, additions, and / or Refers to a polypeptide or peptide sequence that may have substitutions with natural amino acids, non-natural amino acids, or peptidomimetics. In one aspect, a derived sequence shares at least about 40%, 50%, 60%, or 70%, and less than 100% sequence identity with a wild-type or naturally occurring sequence. . In another embodiment, the derivative can be a fragment of a polypeptide, wherein the fragment is at least about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length with a wild-type polypeptide. Are substantially identical (eg, at least about 70%, 75%, 80%, 85%, 90%, or 95% identical). In yet another embodiment, if the polypeptide is attached directly or indirectly to a moiety that is not present in the wild-type polypeptide (eg, a polymer such as PEG), both polypeptides may be amino acids. A polypeptide is “derived from” a wild-type polypeptide even though it shares 100% identity in sequence.

  The natriuretic peptide precursor C (NPPC) polypeptide is a single chain 126 amino acid pre-pro polypeptide and ultimately results in wild type CNP-22 (wtCNP-22) after processing, as described below. . Removal of the signal peptide from NPPC yields pro-CNP and is further cleaved by the endoprotease furin to yield the active 53 amino acid peptide (CNP-53), which is also secreted and cleaved to form a mature 22 amino acid peptide. (CNP or CNP-22) is generated. In one embodiment, a “CNP variant” is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% with wild-type NPPC for the same number of amino acid residues. Or 95% identical.

  The term “effective amount” means a dosage sufficient to produce a desired result for a subject's health, pathology, or disease, or for diagnostic purposes. Desired results may include subjective or objective improvements in dosage recipients. “Therapeutically effective amount” refers to the amount of an agent effective to produce the intended beneficial effect on health. The particular dose level and frequency of dosage for any particular patient may vary and the activity of the particular compound used; bioavailability, metabolic stability, excretion rate of the compound, and It will be appreciated that it will depend on a variety of factors including the length of action; the mode and time of administration of the compound; the age, weight, health status, gender, and diet of the patient; and the severity of a particular symptom. .

  “Treatment” refers to prophylactic treatment or therapeutic treatment or diagnostic treatment. In certain embodiments, “treatment” refers to the administration of a compound or composition to a subject for therapeutic, prophylactic, or diagnostic purposes.

  A “prophylactic” treatment is a treatment administered to a subject who does not show signs of disease, or who shows only early signs of disease, for the purpose of reducing the risk of developing pathology. The compounds or compositions of the present disclosure may be given as a prophylactic treatment to reduce the likelihood of developing the pathology or, if progressed, to minimize the severity of the pathology.

  A “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology with the purpose of attenuating or eliminating the signs or symptoms. Signs or symptoms can be biochemical, cellular, histological, functional, physical, subjective, or It may be objective. The compounds of this disclosure may be given as a therapeutic treatment or for diagnosis.

  “Diagnosis” means identifying the presence, extent, and / or nature of a medical condition. Diagnostic methods differ in their specificity and selectivity. Certain diagnostic methods are unable to make a definitive diagnosis of symptoms, but are sufficient if the method provides a positive indicator to aid diagnosis. A diagnostic procedure will administer a compound of this disclosure to assist in the diagnosis of a subject.

  A “cartilage-related biomarker” or “cartilage-related marker” refers to a growth factor, enzyme, protein, nucleic acid, or other detectable biological material or moiety, and these levels are, for example, cartilage metabolism Increases or decreases in relation to cartilage formation and / or cartilage growth. Such biomarkers may be measured before, during, and / or after administration of the CNP variants described herein. Exemplary cartilage-related biomarkers include, but are not limited to, CNP, cGMP, collagen type II propeptide and fragments thereof, collagen type II and fragments thereof, collagen type I propeptide and fragments thereof, collagen type I and Its fragments, proliferating cell nuclear antigen (PCNA), chondroitin sulfate aggrecan, syndecan-3, and annexin VI. Cartilage-related biomarkers can be measured in any suitable biological sample including, but not limited to, tissue, blood, serum, plasma, spinal fluid, synovial fluid, and urine. In some embodiments, the biomarker is measured in blood, plasma, or serum from an animal that has undergone efficacy / pharmacodynamic in vivo testing and / or from conditioned media from an ex vivo test.

  In certain embodiments, the level of at least one cartilage-related biomarker is measured, and the amount or frequency of administration of the CNP variant administered to the subject can be adjusted according to the level of biomarker measured. In some embodiments, the level of the biomarker is “below the target level” or “above the target level”. The target level of a biomarker is the level or range of levels at which a therapeutic effect is observed in a subject that has received a CNP variant. In certain embodiments, the target level of a biomarker for a subject suffering from osteoarthritis is the level or range of levels of the biomarker observed in a normal, unaffected subject. In various embodiments, the target level of a biomarker need not be equal to the level or range of levels of a biomarker observed in a normal subject to show a therapeutic effect, but is observed, for example, in an unaffected subject. Within 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the “normal” level or range of levels It's okay.

  For example, if the level of the biomarker increases in relation to cartilage formation or growth, the target level of the biomarker that exhibits a therapeutic effect is the biomarker in patients suffering from osteoarthritis who have not been administered the CNP variant. Can be higher than the level of the marker, and optionally can be lower than the “normal” level of the biomarker in a subject not suffering from the disorder, can be about “normal” level, or “normal” level Can exceed. In one aspect, if the biomarker level is below the target level, this indicates an insufficient therapeutic effect and an increase in the amount or frequency of administration of the administered CNP variant may be required. In a related embodiment, if the biomarker is above the target level, this indicates that more CNP variant was administered than needed, and the amount or frequency of administration of the administered CNP variant is reduced. May be needed.

  As another example, if the level of a biomarker decreases in relation to cartilage formation or growth, the target level of a biomarker that exhibits a therapeutic effect suffers from osteoarthritis that was not administered a CNP variant. Can be lower than the level of the biomarker in the patient, and optionally can be higher than the “normal” level of the biomarker in a subject not suffering from the disorder, can be about “normal” level, or “normal” Can be below the “na” level. In such cases, the reverse of previous adjustments in the amount and frequency of administration of the CNP variant may be applied.

  “Pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject animal, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of a CNP variant, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In one aspect, the pharmaceutical composition comprises a composition comprising an active ingredient, and an inactive ingredient that constitutes the carrier, and any two or more combinations, complex formation, or aggregation of the ingredients directly, or Any product resulting from indirectly or due to the separation of one or more of the components or due to one or more other types of reactions or interactions of the components is included. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

  “Pharmaceutically acceptable carrier” refers to standard pharmaceutical carriers, buffers, etc., such as phosphate buffered saline solutions, 5% dextrose solutions, and emulsions (eg, oil / water emulsions or water / oil emulsions). Point to either. Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). The preferred pharmaceutical carrier will depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (eg, oral) or parenteral (eg, subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal). It is done.

  A “pharmaceutically acceptable salt” is a salt that can be incorporated into a compound for pharmaceutical use, including, but not limited to, a metal salt (eg, sodium, potassium, magnesium, calcium, etc.) and ammonia or Contains organic amine salts.

  By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material that is not biologically or otherwise undesirable. That is, the material does not cause any undesired biological effects, or adversely interacts with any of the components of the composition in which it is contained, or with any component present in the individual's body or body. It can be administered to an individual without.

  The term “unit dosage form” refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit calculated as an amount sufficient to produce the desired effect. A predetermined amount of a compound of the present disclosure is optionally included, along with a pharmaceutically acceptable excipient, diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound used and the effect to be achieved and the pharmacodynamics associated with each compound in the host.

  “Physiological condition” refers to a condition in the body of an animal (eg, a human). Physiological conditions include, but are not limited to, body temperature, and physiological ionic strength, pH, and the aqueous environment of the enzyme.

  The term “subject” as used herein includes humans and non-human mammals, as well as non-mammals, to the extent that they are disclosed herein when administered to such non-human species. Range of useful compositions. Examples of mammals include, but are not limited to, mammals: humans, non-human primates such as chimpanzees, and other apes and monkey species; livestock animals such as cattle, horses, sheep, goats, pigs; Examples include any member such as rabbits, dogs, and cats; rodents such as lab animals including rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, vertebrates such as birds and fish. The term does not imply a particular age or gender.

The terms “polyethylene glycol”, “PEG”, “polyethylene oxide”, and “PEO” are used interchangeably herein unless otherwise specified. CNP variants attached via an amino group to a “PEOn” polymer with the number n generally have the formula CH ₃ — [— O—CH ₂ CH ₂ —] _n —C (═O) —NHR. Wherein n is the number of ethylene oxide units and R represents the remaining peptide. The “PEOn” polymer may optionally have an alkylene group, (CH ₂ ) _m , between the carbonyl carbon and the repeating ethylene oxide unit, where m is an integer from 1 to 5. Such “PEOn” (eg, PEO12 or PEO24) polymers are monodisperse, ie, a single discontinuous polymer of a particular molecular weight. Similarly, a CNP variant attached via an amino group to a “PEGnK” polymer with a number nK generally has the formula CH ₃ — [— O—CH ₂ CH ₂ —] _p —C (═O) -NHR, where p is an integer greater than 1. The “PEGnK” polymer may also optionally have an alkylene group, (CH ₂ ) _m , between the carbonyl carbon and the repeating ethylene oxide unit, where m is an integer from 1 to 5. However, such “PEGnK” (eg, PEG1K, PEG2K, PEG5K, or PEG20K) polymers are polydisperse, that is, contain a mixture of polymers having a molecular weight distribution, where the number nK is the number average polymer The molecular weight (M _n ) is expressed in kilodaltons. For example, “PEG2K” attached to a CNP variant represents a polydisperse PEG polymer with a polymer number average molecular weight of approximately 2 kDa.

  Where a mass range of a polymer (eg, PEG) is given (eg, in units of kDa), that range is not the range of molecular weights of the polymers in the polydisperse mixture, but the number of polymers, unless expressly indicated otherwise. Refers to the average molecular weight range.

  The term “halogen”, “halide”, or “halo” refers to fluorine, chlorine, bromine, and / or iodine.

The term “alkyl” refers to a straight-chain or branched saturated monovalent hydrocarbon radical, wherein the alkyl may be substituted with one or more substituents Q as described herein. In certain embodiments, the alkyl is 1 to 20 (C _1-20 ), 1 to 15 (C _1-15 ), 1 to 12 (C _1-12 ), 1 to 10 (C _1-10 ), or 1 Straight chain saturated monovalent hydrocarbon radical having from 6 to (C _1-6 ) carbon atoms, or 3 to 20 (C _3-20 ), 3 to 15 (C _3-15 ), 3 to 12 (C _{3 -12} ) branched saturated monovalent hydrocarbon radicals of 3 to 10 (C _3-10 ), or 3 to 6 (C _3-6 ) carbon atoms. As used herein, straight chain C _1-6 alkyl groups and branched C _3-6 alkyl groups are also referred to as “lower alkyl”. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including all isomeric forms including n-propyl and isopropyl), butyl (n-butyl, isobutyl, sec-butyl, and tert-butyl) Including all isomeric forms), pentyl (including all isomeric forms), and hexyl (including all isomeric forms). For example, C _1-6 alkyl refers to a linear saturated monovalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 6 carbon atoms.

  The term “alkoxy” refers to an —O-alkyl group. In certain embodiments, an alkoxy group may be substituted with one or more substituents Q described herein.

  The term “haloalkyl” refers to an alkyl group substituted with one or more halide atoms. In certain embodiments, the haloalkyl group is substituted with 1, 2, 3, 4, 5, or 6 halide atoms. In certain embodiments, the haloalkyl group may be substituted with one or more additional substituents Q described herein.

The term “cycloalkyl” refers to a cyclic saturated bridged monovalent hydrocarbon radical and / or an unbridged monovalent hydrocarbon radical, which may be substituted with one or more substituents Q as described herein. Point to. In certain embodiments, the cycloalkyl is 3 to 20 (C _3-20 ), 3 to 15 (C _3-15 ), 3 to 12 (C _3-12 ), 3 to 10 (C _3-10 ), or It has 3 to 7 (C _3-7 ) carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl, and adamantyl.

  The term “heterocyclyl” or “heterocyclic” refers to a monocyclic non-aromatic or polycyclic ring system containing at least one non-aromatic ring, wherein one or more of the non-aromatic ring atoms is , O, S, or N are heteroatoms independently selected, and the remaining non-aromatic ring atoms are carbon atoms. In certain embodiments, the heterocyclyl or heterocyclic group has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, or 5 to 6 ring atoms. In certain embodiments, the heterocyclyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems, and a nitrogen or sulfur atom. May be oxidized, the nitrogen atom may be quaternized, and some rings may be partially or fully saturated or aromatic. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom that results in a stable compound. Examples of heterocyclic groups include, but are not limited to, acridinyl, azepinyl, benzimidazolyl, benzindolyl, benzisoxazolyl, benzisoxazinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl, Benzofuranyl, benzonaphthfuranyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiadiazolyl, benzothiazolyl, benzothiophenyl, benzotriazolyl, benzothiopyranyl, benzooxazinyl, benzoxazolyl, Benzothiazolyl, β-carbolinyl, carbazolyl, chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl, dibenzofuranyl, dihydrobenzisothiazinyl, dihydrobenz Soxazinyl, dihydrofuryl, dihydropyranyl, dioxolanyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrazolyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, furanyl, imidazolidinyl, imidazolinyl, imidazolyl, imidazol Pyridinyl, imidazothiazolyl, indazolyl, indolinyl, indolizinyl, indolyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl, isochromanyl, isocoumarinyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isoxax Zolidinyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroindolyl, octa Hydroisoindolyl, oxadiazolyl, oxazolidinyl, oxazolidinyl, oxazolidinyl, oxazolyl, oxiranyl, perimidinyl, phenanthridinyl, phenathrolinyl, phenalsadinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, piperidinyl, 4-peridinyl Piperidonyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyridinyl, pyridopyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuryl, tetrahydrofuranyl, tetrahydroisohydropyranyl, tetrahydroisohydropyranyl Tetrahydrothienyl, tetrazolyl, thiadiazolopi Mijiniru, thiadiazolyl, thiamorpholinyl, thiazolidinyl, thiazolyl, thienyl, triazinyl, triazolyl, and 1,3,5-trithianyl, and the like. In certain embodiments, the heterocyclic group may be substituted with one or more substituents Q described herein.

The term “aryl” refers to a monocyclic aromatic group or polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, the aryl has 6 to 20 (C _6-20 ), 6 to 15 (C _6-15 ), or 6 to 10 (C _6-10 ) ring atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to a bicyclic or tricyclic carbocycle where at least one of the rings is aromatic and the others may be saturated or partially unsaturated. May be aromatic, for example, dihydronaphthyl, indenyl, indanyl, and tetrahydronaphthyl (tetralinyl). In certain embodiments, the aryl group may be substituted with one or more substituents Q described herein.

  The term “heteroaryl” refers to a monocyclic aromatic group or polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring is independently of O, S, and N Contains one or more selected heteroatoms. Each ring of the heteroaryl group may contain 1 or 2 O atoms, 1 or 2 S atoms, and / or 1 to 4 N atoms. However, this is limited to the case where the total number of heteroatoms in each ring is 4 or less, and each ring contains at least one carbon atom. The heteroaryl may be attached to the main structure at any heteroatom or carbon atom that results in a stable compound. In certain embodiments, the heteroaryl has 5 to 20, 5 to 15, or 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and Triazinyl is mentioned. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl Nyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl. In certain embodiments, the heteroaryl group may be substituted with one or more substituents Q described herein.

The term “optionally substituted” means that a group comprising alkyl, alkoxy, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl has one or more substituents Q (in one embodiment, 1, 2, 3 , Or 4 substituents Q) are intended to mean that each Q is cyano, halo, oxo, nitro, C _1-6 alkyl, C _1-6 alkoxy, halo C _{6 alkyl, C 3 to 7} cycloalkyl, _{heterocyclyl, C having 6 to 14} aryl, ^{heteroaryl, -C (O) R e,} -C (O) OR e, -C (O) NR f R g, - C (NR ^e ) NR ^f R ^g , -OR ^e , -OC (O) R ^e , -OC (O) OR ^e , -OC (O) NR ^f R ^g , -OC (= NR ^e ) NR ^f R ^g , -OS (O) R ^{e _{, -OS (O) 2 R e}} , -OS (O) NR f R g, -OS (O) 2 NR f R g, -NR f R g, -NR e C (O) R f, -NR e ^{C (O) OR f, -NR} e C (O) NR f R g, -NR e C (= NR h) NR f R g, -NR e S (O) R f, -NR e S (O) ^{_{2 R f, -NR e S (}} O) NR f R g, -NR e S (O) 2 NR f R g, -SR e, -S (O) R e, -S (O) 2 R e, And —S (O) ₂ NR ^f R ^g , wherein R ^e , R ^f , R ^g , and R ^h are each independently hydrogen, C _1-6 alkyl , _{C 3 to 7} cycloalkyl, _{heterocyclyl,} is a _{C having 6 to 14} aryl or heteroaryl; or ^{R f} and ^{R g,} it take Together with the N atom to which eclipsed, to form a heterocyclyl.

B. CNP variants The use of CNP22 as a therapeutic agent is limited by its short half-life in plasma (J. Clin. Endocrinol. Metab., 78: 1428-35 (1994)). The concentration of CNP22 in human plasma is typically less than 5 picomolar. CNP22 is degraded in humans by NEP and NPR-C and cleared from the circulatory system (Growth Hormone & IGF Res., 16: S6-S14). In all human and animal studies with systemically administered CNP22, continuous infusion has been used to increase CNP22 concentrations in subjects. A CNP peptide with a longer half-life and at least a similar level of functionality would be beneficial for CNP-based therapeutic strategies. CNP variants are also disclosed in related issued patents: US Pat. No. 8,377,884; US Pat. No. 8,198,242; and US Pat. No. 8,598,121. Each of the listed patents is specifically incorporated herein by reference in its entirety.

  The present disclosure has low affinity for NEP and / or NPR-C and low sensitivity to cleavage by NEP and / or clearance by NPR-C, but the functionality is substantially similar to wild-type CNP22 Or a CNP variant that is good. By reducing the sensitivity of the CNP variant to cleavage by NEP and / or clearance by NPR-C, the variant is distributed to the target tissue and site by increasing the plasma half-life or serum half-life of the variant, It is believed that the opportunity to achieve the desired pharmacological effect is increased. In certain embodiments, the CNP variants described herein have an in vitro or in vivo sensitivity to cleavage by NEP and / or clearance by NPR-C of at least about 1.5 compared to wtCNP22. Fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, or 5 fold lower, and in vivo plasma or serum half-life compared to wtCNP22 At least about 50% of the functionality of wtCNP22, while at least about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times increased, Retain 60%, 70%, 80%, 90%, or 100%, or at least about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4% more functional than wtCNP22 Double, 4.5 times, or 5 times larger It was bought. CNP functionality can be assessed for the level of one or more biomarkers (eg, cGMP) associated with cartilage formation or growth, eg, in an in vitro or in vivo test. CNP functionality can also be assessed by determining prevention of arthritic cartilage degeneration, such as in an ex vivo test or in vivo test.

  NEP's natural substrate is small and natriuretic peptides (about 2.2 to about 3.2 kDa) are the largest of NEP's natural substrates. According to X-ray crystallography, the NEP active site is deeply buried inside the central cavity, effectively limiting the size of the substrate molecule to about 3 kDa or less (Oefner et al., J. Mol. Biol., 296: 341-349 (2000)). Based on NPR-B signaling studies, variants of CNP-22, such as CNP-17 (which retains only the cyclic domain of CNP22, Cys6-Cys22), and CNP-53 (31 at the N-terminus) CNP-22) with an amino acid extension can still bind to and activate NPR-B, similar to the 2.2 kDa wtCNP-22. Accordingly, this disclosure includes CNP variants that are attached to natural polymers (eg, peptides) and / or synthetic polymers (eg, PEG) at the N-terminus and / or C-terminus of CNP22 or variants thereof, Show increased NEP resistance but retain the ability to bind to and activate the NPR-B signaling receptor.

によって表されるＣＮＰ変異体を包含し、式中：
（ｘ）および（ｚ）は、それぞれ独立して、天然のポリマー（例えば、少なくとも１つのアミノ酸を含有するペプチド配列）および／または本明細書中に記載される合成ポリマー（例えばＰＥＧ）であり、ＣＮＰ変異体の総質量は、本明細書中に一般に記載される範囲によって特徴付けられ、例えば約２．６ｋＤａまたは２．８ｋＤａから約６または７ｋＤａの範囲である。一態様において、Ｃｙｓ６からＣｙｓ２２の残基は、環状部分を形成する。一態様において、（ｘ）および／または（ｚ）は、ＮＰＰＣポリペプチドまたは非ＣＮＰポリペプチド（例えば、ＡＮＰ、ＢＮＰ、ＩｇＧ等）に由来するアミノ酸伸長を含み、当該伸長は、１から４０、１から３５、１から３１、５から３５、５から３１、または５から１５個のアミノ酸を含有する。別の態様において、ＣＮＰ変異体は、別の天然のアミノ酸、非天然のアミノ酸、ペプチド模倣体、および／またはペプチド結合イソスターによる１つまたは複数の改変および／または置換を、ＣＮＰ２２の以下の位置の１つまたは複数にて含む：ＣＮＰ２２：Ｇｌｙ１、Ｌｙｓ４、Ｇｌｙ５、Ｃｙｓ６、Ｐｈｅ７、Ｇｌｙ８、Ｌｅｕ９、Ｌｙｓ１０、Ｌｅｕ１１、Ｉｌｅ１４、Ｇｌｙ１５、Ｓｅｒ１６、Ｍｅｔ１７、Ｇｌｙ１９、Ｌｅｕ２０、およびＧｌｙ２１。 In one aspect, the disclosure provides a general formula:

Including CNP variants represented by:
(X) and (z) are each independently a natural polymer (eg, a peptide sequence containing at least one amino acid) and / or a synthetic polymer (eg, PEG) described herein; The total mass of the CNP variant is characterized by the ranges generally described herein, for example in the range of about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa. In one aspect, residues Cys6 to Cys22 form a cyclic moiety. In one aspect, (x) and / or (z) comprises an amino acid extension derived from an NPPC polypeptide or a non-CNP polypeptide (eg, ANP, BNP, IgG, etc.), wherein the extension is from 1 to 40, 1 Contains from 35, 1 to 31, 5 to 35, 5 to 31, or 5 to 15 amino acids. In another embodiment, the CNP variant has one or more modifications and / or substitutions with other natural amino acids, non-natural amino acids, peptidomimetics, and / or peptide-bound isosteres at the following positions of CNP22: Including one or more: CNP22: Gly1, Lys4, Gly5, Cys6, Phe7, Gly8, Leu9, Lys10, Leu11, Ile14, Gly15, Ser16, Met17, Gly19, Leu20, and Gly21.

によって表され、式中：
（ｘ）は、合成もしくは天然のポリマー基、またはそれらの組合せであり、合成ポリマー基の非限定的な例として、ＰＥＧ（またはＰＥＯ）があり、天然のポリマー基の非限定的な例として、１から３５個のアミノ酸を含有し、かつＮＰＰＣまたはその、置換および／もしくは欠失を有する変異体、ＡＮＰ、ＢＮＰ、または他の非ＣＮＰ（ポリ）ペプチド、例えば、血清アルブミン、ＩｇＧ、ヒスチジンリッチ糖タンパク質、フィブロネクチン、フィブリノゲン、亜鉛フィンガー含有ポリペプチド、オステオクリン、または線維芽細胞増殖因子２（ＦＧＦ２）に由来するアミノ酸配列があり；
（ｚ）は、不在であってもよいし、合成もしくは天然のポリマー基、またはそれらの組合せであってもよく、合成ポリマー基の非限定的な例として、ＰＥＧがあり、天然ポリマー基の非限定的な例として、ナトリウム利尿ポリペプチド（例えば、ＮＰＰＣ、ＣＮＰ、ＡＮＰ、またはＢＮＰ）、または非ナトリウム利尿ポリペプチド（例えば、血清アルブミンまたはＩｇＧ）に由来するアミノ酸配列があり；かつ
（ｂ）および（ｈ）は、独立して、その位置にて野生型Ｌｙｓであってもよいし、保存的アミノ酸置換基、あるいは以下に限定されないが、Ａｒｇ、Ｇｌｙ、６−ヒドロキシ−ノルロイシン、シトルリン（Ｃｉｔ）、Ｇｌｎ、Ｇｌｕ、またはＳｅｒを含む、任意の天然もしくは非天然のアミノ酸、または側鎖上に反応性第一級アミンを有していないペプチド模倣体で置換されていてもよい。一態様において、（ｂ）はＡｒｇである。別の態様において、ＮＥＰ耐性の向上のために、（ｂ）はＧｌｙでない。 In another aspect, the total mass is characterized by the generally described herein, for example, in the range of about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa, such that resistance to NEP degradation is increased. The designed CNP variant has the general formula:

Represented by the formula:
(X) is a synthetic or natural polymer group, or a combination thereof, a non-limiting example of a synthetic polymer group is PEG (or PEO), and a non-limiting example of a natural polymer group is Variants, ANP, BNP, or other non-CNP (poly) peptides containing 1 to 35 amino acids and having substitutions and / or deletions thereof, such as serum albumin, IgG, histidine rich sugars There is an amino acid sequence derived from a protein, fibronectin, fibrinogen, zinc finger-containing polypeptide, osteocrine, or fibroblast growth factor 2 (FGF2);
(Z) may be absent, may be a synthetic or natural polymer group, or a combination thereof, a non-limiting example of a synthetic polymer group is PEG, Limited examples include amino acid sequences derived from natriuretic polypeptides (eg, NPPC, CNP, ANP, or BNP), or non-natriuretic polypeptides (eg, serum albumin or IgG); and (b) and (H) may independently be wild-type Lys at that position, conservative amino acid substituent, or, but not limited to, Arg, Gly, 6-hydroxy-norleucine, citrulline (Cit) Any natural or non-natural amino acid, or reactive primary amine on the side chain, including, Gln, Glu, or Ser It may be substituted with peptidomimetic that does not have. In one embodiment, (b) is Arg. In another embodiment, (b) is not Gly for improved NEP resistance.

非ＣＮＰポリペプチド、例えば、ＡＮＰ、ＢＮＰ、血清アルブミン、およびＩｇＧに由来するアミノ酸配列の非限定的な例として、以下が挙げられる：

In yet another embodiment, (h) is not Arg.
Non-limiting examples of amino acid sequences derived from NPPC or variants thereof include the following:

Non-limiting examples of amino acid sequences derived from non-CNP polypeptides such as ANP, BNP, serum albumin, and IgG include the following:

  In one aspect, any N-terminal (x) group and / or C-terminal (z) group of a CNP variant having a (x) group and / or a (z) group described herein is Independently, it includes an amino acid sequence containing a small number of acidic natural or non-natural amino acids, if any (eg, Asp or Glu). In another embodiment, (x) and / or (z) is a basic natural or non-natural amino acid (eg, Lys, Arg) in order to maintain an alkaline pI comparable to that of CNP22 (pI 8.9). Or His) is enriched. In one aspect, the pI of the CNP variant is in the range of about 8 to about 10.5, such that the CNP variant diffuses more easily through the extracellular matrix surrounding the arthritic joint chondrocytes. Designed to be able to. In more stringent embodiments, the pI of the CNP variant is from about 8.5 to about 10.5, or from about 8.5 to about 10, or from about 9 to about 10.

  In yet another embodiment, (x) and / or (z) are enriched with polar natural or non-natural amino acids and are designed to increase aqueous solubility. In yet another embodiment, (x) and / or (z) contains a small number of hydrophobic natural or non-natural amino acids, if any (eg, Ala, Val, Leu, Ile, or Met). .

  In a further embodiment, the N-terminus of the CNP variant ends with at least one glycine residue and is designed to increase serum half-life. In a related embodiment, to prevent pyroglutamine formation, the N-terminus of a CNP variant that normally ends with glutamine ends with a glycine residue. In one embodiment, the (x) group contains an amino acid extension that ends at the N-terminus with at least one glycine residue. In another embodiment, (x) and / or (z) does not contain two adjacent basic natural or non-natural amino acids (eg, Lys-Lys or Arg-Arg) and is against cleavage by protease furin. Designed to reduce sensitivity. In one aspect, (x) does not contain two adjacent basic amino acids immediately before the position corresponding to Gly1 of CNP22.

  In yet another embodiment, the (x) group and / or (z) group of the CNP variant comprises an amino acid sequence derived from NPPC (eg, derived from CNP53). In one aspect, (x) comprises an amino acid sequence derived from the N-terminal tail of ANP or BNP. In another embodiment, (z) comprises an amino acid sequence derived from the C-terminal tail of ANP or BNP. In further embodiments, (x) and / or (z) is a non-natriuretic polypeptide, such as IgG, human serum albumin (HSA), histidine-rich glycoprotein, fibronectin, fibrinogen, zinc finger-containing polypeptide, FGF- 2, and amino acid sequences derived from bone target proteins (eg, osteoocrine, osteopontin, osteocalcin, and sialoprotein).

  In any embodiment described herein wherein CNP22 or a variant thereof may have an N-terminal (x) group and / or a C-terminal (z) group, (x) and / or (z) May independently contain an amino acid sequence derived from a functional domain of a bone morphogenetic protein (BMP). An N-terminal and / or C-terminal amino acid extension derived from the functional domain of BMP will reduce the total mass of the CNP variant to the range generally described herein, eg, from about 2.6 kDa or 2.8 kDa to about By increasing it to be characterized in the 6 or 7 kDa range, it is possible to increase the NEP resistance of the CNP variant and hence the serum half-life. In addition, since certain BMPs are growth factors and cytokines that induce bone and cartilage formation, a fragment derived from the functional domain of BMP is guanylyl cyclase of NPR-B by the cyclic domain of CNP22 or a variant thereof. Mechanisms different from functional activation can promote chondrocyte, cartilage, or bone growth. Non-limiting examples of BMPs that promote bone formation and development, cartilage formation and development, and / or osteoblast differentiation include BMP1, BMP2, BMP3, BMP5, BMP7, and BMP8a. In one embodiment, the N-terminus and / or C-terminus of CNP22 or a variant thereof is independently an amino acid derived from the last 140 amino acids in the C-terminal portion of BMP1, BMP2, BMP3, BMP5, BMP7, or BMP8a Bound to an array.

を含むアミノ酸伸長を含有する。 In one aspect, the CNP variant is not limited to the following at the N-terminus and / or C-terminus of CNP22 or CNP17,

Containing amino acid extensions.

In another embodiment, the CNP variant has a K4R substitution at position 4 of CNP22. Non-limiting examples of CNP (K4R) variants include the following:

  In a further embodiment, the CNP variant is CNP22 or a variant thereof having amino acid additions, deletions, and / or substitutions, and a peptide fragment derived from a polypeptide or protein other than CNP relative to the N-terminus of the CNP peptide. Or a chimera comprising the entire non-CNP polypeptide or protein, and CNP22 or a variant thereof may have an N-terminal amino acid extension of one or more amino acid residues. In certain embodiments, the CNP chimera comprises CNP22 or a variant thereof having an N-terminal amino acid extension of one or more amino acid residues. In certain embodiments, the CNP chimera contains a lysine-lysine (KK) or GANKK residue immediately prior to the first position of CNP22 or a variant thereof (Gly in the case of CNP22). In other embodiments, the CNP chimera contains one or two residues that differ from lysine-lysine immediately prior to the first position of CNP22 or a variant thereof. Non-limiting examples of residues that can immediately precede the first position of CNP22 or variants thereof include KP, PK, PR, PQ, QK, QQ, RR, SS, GANKP, GANPK, GANPR, GANPQ, GANQK , GANQQ, GANRR, and GANSS.

を含む。 In another aspect, the CNP variant is a chimera comprising CNP22 and an N-terminal peptide fragment, including but not limited to:

including.

を含む。 In yet another aspect, the CNP variant is a chimera comprising an N-terminal peptide fragment and CNP22 (“CNP22 (K4R)”) in which arginine is replaced with Lys4 of CNP22, including but not limited to:

including.

  Chimeras comprising IgG and CNP22 or variants thereof are specifically designed for increased resistance to NEP degradation and decreased binding to serum albumin. CNP chimeras containing surface fragments of HSA are specifically designed for reduced immunogenicity and reduced binding to serum albumin. HRGP-CNP22 and HRGP-CNP22 (K4R) chimeras containing cationic, histidine-rich, non-lysine, non-arginine sequences at the N-terminus are specifically designed for increased stability against proteases. Chimeras containing osteocrine fragments are designed to release osteoocrine fragments in the bone growth plate immediately after protease (eg, furin) cleavage, where the fragments are thought to inhibit the clearance receptor NPR-C. It is done. For chimeras containing FGF2 heparin binding fragments, heparin binding to the fragments is designed to achieve a longer serum half-life by protecting the chimera from degradation. Chimeras containing fibronectin, fibrinogen, or zinc finger fragments have been designed for reduced binding to serum albumin, among other features.

  While not intending to be bound by theory, it has increased resistance to NEP degradation and functionalities (eg, binding to NPR-B and stimulation of cGMP signaling) compared to wtCNP22. CNP variants with a molecular weight of about 2.6 or 2.8 kDa to about 6 or 7 kDa may be more effective if they do not bind tightly to plasma proteins such as serum albumin. CNP variants that do not bind tightly to plasma proteins (eg, serum albumin) diffuse through the cartilage and arrive at the articular chondrocytes and bind to NPR-B for cGMP signaling. May be more effective in activating. In one aspect, a CNP variant designed for reduced binding to plasma proteins (eg, serum albumin) is a chimera comprising CNP22 or a variant thereof and a peptide fragment derived from IgG. In another aspect, CNP variants designed for reduced binding to plasma proteins include CNP22 or CNP22 (K4R) and polypeptides (eg, IgG, HSA, fibronectin, fibrinogen, zinc finger-containing polypeptides, etc. A chimera containing a fragment derived from In yet another aspect, a CNP variant designed for reduced binding to plasma proteins comprises CNP22 or a variant thereof conjugated to a hydrophilic or water soluble polymer. In one embodiment, the hydrophilic or water soluble polymer is PEG (or PEO). In another embodiment, the hydrophilic or water-soluble polymer (eg, PEG) is one or more functional groups that impart a negative charge to the polymer under physiological conditions, such as carboxyl groups, sulfate groups, or phosphates. Functionalized with groups, or combinations thereof.

In a further aspect, the CNP variants of the present disclosure include truncated forms having a wild-type amino acid sequence from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53) and derived from hCNP-53. (Truncated) CNP peptide. Such truncated CNP peptides include the following:

  In certain embodiments, the CNP variant does not comprise CNP-17, CNP-22, or CNP-53.

In another aspect, a truncated CNP peptide ranging from hCNP-17 to hCNP-53 is an amino acid sequence described herein at any one or more of the amino acid positions of a particular truncated CNP peptide. Additions, deletions, and / or substitutions with natural or non-natural amino acids or peptidomimetics (eg, peptide-bound isosteres) may be included. In yet another embodiment, a truncated CNP peptide having a wild-type sequence or amino acid addition, deletion, and / or substitution is herein described at the N-terminus, C-terminus, and / or internal site. May be attached to any of the described moieties, including but not limited to synovial targeting moiety or cartilage targeting moiety (eg, bisphosphonate, bone targeting peptide sequence or cartilage targeting peptide sequence (eg, polyAsp, Poly Glu), peptide sequences derived from bone target domains of bone proteins (eg, osteopontin, osteocalcin, sialoprotein), peptides derived from functional domains of bone morphogenetic proteins (eg, BMP2, BMP3, BMP5, BMP7, BMP8a) Sequence, natriuretic polypeptide (eg, NPP , ANP, BNP), derived from non-natriuretic polypeptide (eg, serum albumin, IgG, histidine-rich glycoprotein, fibronectin, fibrinogen, zinc finger-containing polypeptide, FGF-2, osteocrine) Peptide sequences that reduce renal clearance (eg, negatively charged PEG moieties), hydrophilic polymers (eg, PEG), carbohydrates (eg, carbohydrates recognized by receptors on the surface of cells in arthritic joints) ), hydrophobic acids _(e.g., C 5 _{-C 12} carboxylic acid, natural fatty acids), phospholipids, and combinations thereof. In one embodiment, it has a wild-type sequence, or amino acid additions, deletions, and / or substitutions, and optionally binds to one or more moieties at the N-terminus, C-terminus, and / or internal site. The truncated CNP peptides that have been described are characterized by a total mass generally described herein, for example in the range of about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa.

である。ＣＮＰ３７変異体は、ＣＮＰ３７の３７の位置の任意の１つまたは複数にて、アミノ酸の付加、欠失、および／または天然もしくは非天然のアミノ酸もしくはペプチド模倣体（例えばペプチド結合イソスター）との置換を含有してよい。ＣＮＰ２２のナンバリングに基づいて、ＣＮＰ３７においてなされ得る置換の非限定的な例として、Ｋ４Ｒ、Ｇ５Ｓ、ＧＳＲ、Ｇ８Ｓ、Ｋ１０Ｒ、Ｇ１５Ｓ、Ｓ１６Ｑ、Ｍ１７Ｎ、Ｇ１９Ｒ、およびそれらの組合せが挙げられる。一態様において、ＣＮＰ３７誘導体は、Ｍｅｔ１７の、天然（例えばアスパラギン）または非天然のアミノ酸またはペプチド模倣体への置換を含有し、部分的にメチオニンの硫黄原子の酸化を回避するように設計されている。別の態様において、ＣＮＰ３７変異体は、Ｌｙｓ８、Ｌｙｓ１０、Ｌｙｓ１４、および／またはＬｙｓ１５（ＣＮＰ３７のＮ末端からのナンバリングに基づく）の、非塩基性の天然または非天然のアミノ酸またはペプチド模倣体への置換を含有し、部分的にアルブミン結合を減少させるように設計されている。 In a further embodiment, the CNP variant is a derivative of CNP37, which is

It is. A CNP37 variant is an amino acid addition, deletion, and / or substitution with a natural or non-natural amino acid or peptidomimetic (eg, a peptide-linked isostere) at any one or more of 37 positions of CNP37. May be included. Based on CNP22 numbering, non-limiting examples of substitutions that can be made in CNP37 include K4R, G5S, GSR, G8S, K10R, G15S, S16Q, M17N, G19R, and combinations thereof. In one aspect, the CNP37 derivative contains a substitution of Met17 with a natural (eg, asparagine) or non-natural amino acid or peptidomimetic and is designed to partially avoid oxidation of the sulfur atom of methionine. . In another embodiment, the CNP37 variant is a substitution of Lys8, Lys10, Lys14, and / or Lys15 (based on numbering from the N-terminus of CNP37) to an abasic natural or non-natural amino acid or peptidomimetic And is designed to partially reduce albumin binding.

In addition to or in place of amino acid additions, deletions, and / or substitutions, CNP37 derivatives may include, but are not limited to, bone targeting moieties or N-terminal, C-terminal, and / or internal sites Cartilage targeting moiety (eg, bone targeting peptide domain), moiety that decreases renal clearance (eg, negatively charged PEG moiety), hydrophilic polymer (eg, PEG), amino acid sequence comprising one or more amino acids (eg, osteo) Klin “NPR-C inhibitor” fragment), carbohydrates (eg, carbohydrates recognized by receptors on the surface of bone growth plate cells), hydrophobic acids (eg, C ₅ -C ₁₂ carboxylic acids and natural fatty acids) ), And combinations thereof, may be attached to any of the moieties described herein.

が挙げられる。 In one aspect, the CNP variant is designed to have a mutation / substitution at the furin cleavage site (underlined) to improve in vivo resistance to furin protease and / or N A modified CNP37 peptide designed to contain glycine at the terminus (underlined) to improve plasma stability and prevent pyroglutamine formation. Such CNP37 variants include, but are not limited to:

Is mentioned.

In further embodiments, CNP variants of the present disclosure include CNP peptides and variants thereof that can be produced by the fusion protein process described herein. Non-limiting examples of CNP variants that can be produced by the fusion protein process described herein using chemical or proteolytic cleavage or protein self-cleavage include the following:

  Unless the intended site of chemical or proteolytic cleavage of the fusion protein is present in the amino acid sequence of the target CNP variant itself, other CNP variants (hCNP-17 to hCNP-53 and wild) (Including truncated CNP peptides with type sequences or amino acid additions, deletions and / or substitutions) can also be generated by the fusion protein process described herein. As a non-limiting example, the fusion protein process described herein can be used to generate truncated wtCNP34 using formic acid cleavage.

  In additional embodiments, for any of the CNP peptides and variants described herein having asparagine (Asn / N) and / or glutamine (Gln / Q) residues, these are wild Any Asn residue and / or any Gln residue, independently having a type sequence or non-natural amino acid sequence, independently includes any other conservative substitution, such as Asn to Gln. It may be substituted with a natural or non-natural amino acid. Such substitutions are designed in part to minimize or avoid any possible deamidation of asparagine and / or glutamine. Any Asn residue and / or any Gln residue may be independently substituted with any other natural or non-natural amino acid (including conservative substitutions such as Asn to Gln), CNP peptides and Non-limiting examples of CNP variants include wtCNP34, wtCNP37, Gly-wtCNP37, Pro-wtCNP37, Pro-Gly-wtCNP37, GHKSEVAHRKK-wtCNP27 (SEQ ID NO: 48), Pro-GHKSEVAHRFK-wtCNP27 (SEQ ID NO: 48) 50), PEO12-GANRR-CNP22 (K4R) (SEQ ID NO: 101), and PEO24-GANRR-CNP22 (K4R) (SEQ ID NO: 102). In certain embodiments, the asparagine residues of the CNP peptides and CNP variants described herein are not substituted with glutamine, aspartic acid, or glutamic acid. In certain embodiments, the glutamine residues of the CNP peptides and CNP variants described herein are not substituted with asparagine, aspartic acid, or glutamic acid.

のアスパラギン残基７および／または１５は独立して、アスパラギン残基の、アスパラギン酸またはイソアスパラギン酸への可能性があるいかなる脱アミドも回避するために、グルタミンを含む任意の他の天然または非天然のアミノ酸で置換されていてよい。特定の態様において、Ｐｒｏ−Ｇｌｙ−ｗｔＣＮＰ３７のアスパラギン残基７および／または１５は、グルタミン、アスパラギン酸、またはグルタミン酸で置換されていない。 As a non-limiting example, Pro-Gly-wtCNP37

Asparagine residues 7 and / or 15 independently of any other natural or non-containing, including glutamine, to avoid any possible deamidation of the asparagine residue to aspartic acid or isoaspartic acid. It may be substituted with a natural amino acid. In certain embodiments, asparagine residues 7 and / or 15 of Pro-Gly-wtCNP37 are not substituted with glutamine, aspartic acid, or glutamic acid.

  However, the present disclosure contemplates that any one or more, up to all residues sensitive to deamidation or deamidation-like reactions (eg isomerization) are converted via deamidation or deamidation-like reactions. It is understood to encompass CNP variants that may be converted to other residues to any degree, up to 100% conversion per residue. In certain embodiments, the present disclosure provides: (1) any one or more, up to all asparagine (Asn / N) residues, via deamidation, to aspartic acid or aspartate and / or isoasparagine. May be converted to acid or isoaspartate, the conversion being up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, (2) any one or more, up to all glutamine (Gln / Q) residues, via deamidation, to glutamic acid or glutamate and / or isoglutamic acid or iso May be converted to glutamate, the conversion being up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% per residue converted (3) any one or more, up to all aspartic acid or aspartate (Asp / D) residues undergo deamidation-like reaction (also called isomerization) May be converted to isoaspartic acid or isoaspartate, the conversion being up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70 per converted residue. %, 80%, 90%, or 100%; (4) any one or more, up to all glutamic acid or glutamate (Glu / E) residues undergo a deamidation-like reaction (also called isomerization) May be converted to isoglutamic acid or isoglutamate, the conversion being up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% per converted residue 80%, 90%, or 100%; or (5) above things are combined, it encompasses CNP variants.

の任意の１つまたは複数、最大全てのアスパラギン、グルタミン、アスパラギン酸、および／またはグルタミン酸残基が、先に記載されるように、脱アミドまたは脱アミド様反応を介して、（１）アスパラギン酸／アスパルテートおよび／もしくはイソアスパラギン酸／イソアスパルテート、（２）グルタミン酸／グルタメートおよび／もしくはイソグルタミン酸／イソグルタメート、（３）イソアスパラギン酸／イソアスパルテート、ならびに／または（４）イソグルタミン酸／イソグルタメートにそれぞれ変換されていてよく、変換は、変換される残基あたり最大約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または１００％である、ＣＮＰ変異体を包含する。 As a non-limiting example, the present disclosure provides Pro-Gly-wtCNP37

Any one or more, up to all asparagine, glutamine, aspartic acid, and / or glutamic acid residues may be (1) aspartic acid via a deamidation or deamidation-like reaction, as described above. / Aspartate and / or isoaspartic acid / isoaspartate, (2) glutamic acid / glutamate and / or isoglutamic acid / isoglutamate, (3) isoaspartic acid / isoaspartate, and / or (4) isoglutamic acid / iso Each may be converted to glutamate, the conversion being up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, Or CNP variants that are 100%.

の任意の１つまたは複数、最大全てのアスパラギンおよび／またはアスパラギン酸残基が、脱アミドまたは脱アミド様反応を介して、（１）アスパラギン酸／アスパルテートおよび／もしくはイソアスパラギン酸／イソアスパルテート、ならびに／または（２）イソアスパラギン酸／イソアスパルテートにそれぞれ変換されていてよく、変換は、変換される残基あたり最大約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または１００％である、ＣＮＰ変異体を包含する。 As a further example, the present disclosure provides Pro-Gly-wtCNP37

Any one or more, and at most all asparagine and / or aspartic acid residues, via deamidation or deamidation-like reactions, (1) aspartic acid / aspartate and / or isoaspartic acid / isoaspartate And / or (2) may be converted to isoaspartic acid / isoaspartate, respectively, the conversion being up to about 5%, 10%, 20%, 30%, 40%, 50% per converted residue , 60%, 70%, 80%, 90%, or 100%.

の任意の１つまたは複数、最大全てのアスパラギン、グルタミン、アスパラギン酸、および／またはグルタミン酸残基が、脱アミドまたは脱アミド様反応を介して、（１）アスパラギン酸／アスパルテートおよび／もしくはイソアスパラギン酸／イソアスパルテート、（２）グルタミン酸／グルタメートおよび／もしくはイソグルタミン酸／イソグルタメート、（３）イソアスパラギン酸／イソアスパルテート、ならびに／または（４）イソグルタミン酸／イソグルタメートにそれぞれ変換されていてよく、変換は、変換される残基あたり最大約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または１００％である、ＣＮＰ変異体を包含する。 As another example, the present disclosure provides Gly-wtCNP37

Any one or more, up to all asparagine, glutamine, aspartic acid, and / or glutamic acid residues, via deamidation or deamidation-like reactions, (1) aspartic acid / aspartate and / or isoasparagine Acid / isoaspartate, (2) glutamic acid / glutamate and / or isoglutamic acid / isoglutamate, (3) isoaspartic acid / isoaspartate, and / or (4) may be converted to isoglutamic acid / isoglutamate, respectively. , CNP mutations that are up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per residue to be converted Includes the body.

の任意の１つまたは複数、最大全てのアスパラギン、グルタミン、アスパラギン酸、および／またはグルタミン酸残基が、脱アミドまたは脱アミド様反応を介して、（１）アスパラギン酸／アスパルテートおよび／もしくはイソアスパラギン酸／イソアスパルテート、（２）グルタミン酸／グルタメートおよび／もしくはイソグルタミン酸／イソグルタメート、（３）イソアスパラギン酸／イソアスパルテート、ならびに／または（４）イソグルタミン酸／イソグルタメートにそれぞれ変換されていてよく、変換は、変換される残基あたり最大約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または１００％である、ＣＮＰ変異体を包含する。 As yet another example, the present disclosure provides wtCNP37

Any one or more, up to all asparagine, glutamine, aspartic acid, and / or glutamic acid residues, via deamidation or deamidation-like reactions, (1) aspartic acid / aspartate and / or isoasparagine Acid / isoaspartate, (2) glutamic acid / glutamate and / or isoglutamic acid / isoglutamate, (3) isoaspartic acid / isoaspartate, and / or (4) may be converted to isoglutamic acid / isoglutamate, respectively. , CNP mutations that are up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per residue to be converted Includes the body.

の任意の１つまたは複数、最大全てのアスパラギン、アスパラギン酸、および／またはグルタミン酸残基が、脱アミドまたは脱アミド様反応を介して、（１）アスパラギン酸／アスパルテートおよび／もしくはイソアスパラギン酸／イソアスパルテート、（２）イソアスパラギン酸／イソアスパルテート、ならびに／または（３）イソグルタミン酸／イソグルタメートにそれぞれ変換されていてよく、変換は、変換される残基あたり最大約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または１００％である、ＣＮＰ変異体を包含する。 As a further example, the present disclosure provides an HSA-wtCNP27 chimera,

Any one or more, at most all asparagine, aspartic acid, and / or glutamic acid residues, via deamidation or deamidation-like reactions, (1) aspartic acid / aspartate and / or isoaspartic acid / May be converted to isoaspartate, (2) isoaspartic acid / isoaspartate, and / or (3) isoglutamic acid / isoglutamate, respectively, with conversion up to about 5%, 10% per residue converted , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

の任意の１つまたは複数、最大全てのアスパラギン、アスパラギン酸、および／またはグルタミン酸残基が、脱アミドまたは脱アミド様反応を介して、（１）アスパラギン酸／アスパルテートおよび／もしくはイソアスパラギン酸／イソアスパルテート、（２）イソアスパラギン酸／イソアスパルテート、ならびに／または（３）イソグルタミン酸／イソグルタメートにそれぞれ変換されていてよく、変換は、変換される残基あたり最大約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または１００％である、ＣＮＰ変異体を包含する。 As a further example, the present disclosure provides a Pro-HSA-wtCNP27 chimera,

Any one or more, at most all asparagine, aspartic acid, and / or glutamic acid residues, via deamidation or deamidation-like reactions, (1) aspartic acid / aspartate and / or isoaspartic acid / May be converted to isoaspartate, (2) isoaspartic acid / isoaspartate, and / or (3) isoglutamic acid / isoglutamate, respectively, with conversion up to about 5%, 10% per residue converted , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

  Also, the present disclosure provides that any one or more and at most all methionine (Met / M) residues are oxidized to any chemically viable oxidized form (eg, sulfoxide and / or sulfone). Well, the conversion is up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per oxidized residue, CNP Includes variants.

  In another embodiment, the CNP variant comprises CNP22 or a variant thereof linked at the N-terminus and / or C-terminus to a moiety that promotes the translocation of the variant throughout the cell membrane or cell barrier. In one embodiment, the CNP variant is bound to a peptide sequence that facilitates transport of the variant across the cell membrane or cell barrier (including via an active peptide transporter) at the N-terminus and / or C-terminus. .

  In further embodiments, the N-terminus and / or C-terminus of CNP22 or a variant thereof is linked to a chemical moiety, eg, a natural polymer and / or a synthetic polymer, so that the total mass of the modified CNP peptide is herein Increasing from the generally described range, eg, about 2.6 or 2.8 kDa to about 6 or 7 kDa. In one aspect, the chemical moiety is a biocompatible, hydrophilic or water-soluble natural polymer (eg, peptide, carbohydrate) or synthetic polymer (eg, PEG (or PEO)).

  In certain embodiments, the N-terminus and / or C-terminus of CNP22 or a variant thereof is attached to a PEG (or PEO) polymer and is generally described herein, for example, about 2.6 or 2.8 kDa. To a total mass characterized by a range of about 6 or 7 kDa. PEGylation of CNP22 or variants thereof has been designed to reduce immunogenicity and improve half-life by inter alia reducing renal clearance and increasing protease resistance. The PEG moiety is CNP22 or, but is not limited to, CNP-17 (Cys6-Cys22 cyclized part of CNP22), CNP37, and N-terminal and / or C-terminal amino acid extensions, amino acid substitutions, and / or amino acid deletions It may be attached to the N-terminus and / or C-terminus of any variant described herein, including mutants of CNP17, CNP22, or CNP37 that have a loss. In one aspect, the Lys4 and / or Lys10 residues of CNP17, CNP22, or CNP37, or variants thereof are natural or non-natural amino acids (eg, Arg, Gly, Ser, Gln, Glu, or Cit) Or substituted with peptidomimetics that do not contain a reactive primary amine on the side chain, eliminating any possible pegylation of these lysine residues. In one embodiment, the Lys4 and / or Lys10 residues of the CNP peptide are substituted with Arg. In another embodiment, the Lys10 residue is not substituted with Arg.

  In a further embodiment, CNP variants (including CNP22 and variants thereof) having a PEG (or PEO) moiety and amino acid extension at the N-terminus contain arginine at a position immediately before the position corresponding to Gly1 of CNP22. . Such pegylated CNP variants are designed for increased resistance to NEP degradation, decreased binding to serum albumin, and enhanced CNP functional activity (eg, activation of cGMP signaling). Non-limiting examples of pegylated CNP variants include PEO24-GANRR-CNP22 (K4R) (SEQ ID NO: 102), PEO12-GANRR-CNP22 (K4R) (SEQ ID NO: 101), PEO24-GANRR-CNP22. (SEQ ID NO: 103), PEO12-GANRR-CNP22 (SEQ ID NO: 104), PEO24-GANPR-CNP22 (K4R) (SEQ ID NO: 105), PEO12-GANPR-CNP22 (K4R) (SEQ ID NO: 106), PEO24-GANPR-CNP22 (SEQ ID NO: 107), PEO12-GANPR-CNP22 (SEQ ID NO: 108), PEO24-GANQQ-CNP22 (SEQ ID NO: 109), PE 12-GANQQ-CNP22 (SEQ ID NO: 110), PEO24-ER-CNP22 (K4R) (SEQ ID NO: 111), PEO12-ER-CNP22 (K4R) (SEQ ID NO: 112), PEO24-ER-CNP22 (SEQ ID NO: 113), PEO12-ER-CNP22 (SEQ ID NO: 114), PEO24-R-CNP22 (K4R) (SEQ ID NO: 115), PEO12-R-CNP22 (K4R) (SEQ ID NO: 116), PEO24-R-CNP22 (SEQ ID NO: 117), and PEO12-R-CNP22 (SEQ ID NO: 118), where PEO24 is a monodisperse 1.2 kDa PEG polymer and PEO12 Is monodisperse 0. kDa which is the PEG polymer. In one embodiment, the PEG (or PEO) polymer is attached to the N-terminus of the CNP variant.

  The present disclosure provides for types (eg, homopolymers or copolymers; random copolymers, alternating copolymers, or block copolymers; linear or branched; monodisperse or polydisperse), bonds (eg, hydrolyzable bonds or stable) Bonds, such as amide, imine, aminal, alkylene, or ester bonds), binding sites (eg, N-terminal and / or C-terminal, preferably CNP cyclization region (residues 6-22 in CNP22) Corresponding residues) and lengths (eg, from about 0.2, 0.4, or 0.6 kDa to about 2, 3, 4, or 5 kDa) or hydrophilic polymers or water-soluble The use of a functional polymer (eg PEG) is contemplated. The hydrophilic polymer or water-soluble polymer may be bound to the CNP peptide by N-hydroxysuccinimide (NHS) -based or aldehyde-based chemicals known in the art, or other chemicals. Such CNP variants are, for example, wt CNP22 (2.2 kDa), an amino acid extension at the N-terminus and / or C-terminus of CNP17, CNP22 or CNP17 that retains only the cyclized region of residues CRT22 (residues 6-22) Or mutants having amino acid substitutions, additions and / or deletions, such as GANRR-CNP22 (K4R) (SEQ ID NO: 119), GANPR-CNP22 (K4R) (SEQ ID NO: 78), R-CNP22 (SEQ ID NO: 71), R-CNP22 (K4R) (SEQ ID NO: 79), ER-CNP22 (SEQ ID NO: 72), and ER-CNP22 (K4R) (SEQ ID NO : 80). In one embodiment, a PEG-CNP variant whose total mass is characterized by a range generally described herein, for example, from about 2.6 or 2.8 kDa to about 6 or 7 kDa, is N-terminal and / or Monodispersed linear PEG (or PEO) moiety bound to C-terminus via NHS or aldehyde-based chemicals, or N-terminal and / or C-terminal bound via NHS-based chemicals 2 Contains a branched PEG moiety of the arm or three arms. The present disclosure also includes negatively charged PEG-CNP variants designed for reduced renal clearance, including but not limited to carboxylated compounds, sulfated compounds, and phosphorus Contains oxidized compounds (Caliceti, Adv. Drug Deliv. Rev., 55: 1261-77 (2003); Perlman, J. Clin. Endo. Metab., 88: 3227-35 (2003); Pitkin, Antimicrob. Ag. Chemo., 29: 440-444 (1986); Vehaskari, Kidney Intl, 22: 127-135 (1982)). In one embodiment, the PEG (or PEO) moiety contains a carboxyl group, a sulfate group, and / or a phosphate group.

  In another aspect, the PEG (or PEO) moiety attached to the N-terminus, C-terminus, and / or internal site of the CNP variants described herein is positively charged under physiological conditions. Contains one or more functional groups. Such PEG moieties are specifically designed to improve the distribution of such PEGylated CNP variants into cartilage tissue. In one embodiment, such PEG moieties contain one or more primary amino groups, secondary amino groups, or tertiary amino groups, quaternary ammonium groups, and / or other amine-containing (eg, urea) groups. contains.

In one aspect, the disclosure includes CNP22 or a variant thereof conjugated to a PEG (or PEO) of formula (CH ₂ CH ₂ O) _n via an NHS or aldehyde chemical, wherein , N is an integer from about 6 to about 100, and the PEG polymer is from about 0.3 kDa to about 5 kDa. In another embodiment, n is an integer from about 12 to about 50 and the PEG polymer is from about 0.6 kDa to about 2.5 kDa. In yet another aspect, n is from about 12 to about 24 and the PEG polymer is from about 0.6 kDa to about 1.2 kDa. In yet another embodiment, the terminal hydroxyl groups of the PEG polymer are capped with non-reactive groups. In certain embodiments, the end-capping group is an alkyl group, for example a lower alkyl group such as methyl.

  In a further aspect, the present disclosure provides CNP variants having one or more peptide bonds or peptide bond isosteres that have reduced sensitivity to cleavage by peptidases, including neutral endopeptidase (NEP). NEP is a membrane-bound zinc-dependent endopeptidase that cleaves substrate peptide bonds at the amino terminus of large hydrophobic residues. Thus, modification of a peptide bond at the NEP cleavage site to a non-natural peptide or non-peptide bond can eliminate or reduce the efficiency of NEP cleavage.

  It has been reported for ANP and CNP that NEP cleavage occurs first at the Cys6-Phe7 bond in the cyclization region and then elsewhere throughout the rest of the structure. For BNP, cleavage is reported to occur first at the peptide N-terminus and then within the cyclic structure. The first NEP cleavage site on CNP has been reported to be Cys6-Phe7 binding, but all possible sites were unexpected when wtCNP22 was exposed to NEP digestion in vitro for 2.5 minutes. The Cys6-Phe7 peptide bond and the Gly8-Leu9 peptide bond were slight but the most unstable.

  The substrate specificity of NEP is mainly determined by two substrate-binding subsites, S1 'and S2' (Oefner et al., J. Mol. Biol. 296: 341-349 (2000)). The S1 'site accepts large hydrophobic P1' residues (eg, Phe, Leu, Ile, and Met) where the N-terminal peptide bond is exposed to hydrolysis. The S2 'site generally prefers smaller residues called P2' (eg, Gly or Ser). In the case of CNP, Phe7 has been reported to be the preferred P1 'residue for the NEP S1' site, while Gly8 is the preferred P2 'residue for the S2' site. Since these two subsites can only accommodate a specific total side chain size, any increase in the total size of the CNP P1′-P2 ′ residues potentially disrupts NEP binding. be able to. For example, addition of a chloride atom at the 3-position of the P1′Phe7 aromatic ring (ie, 3-Cl-Phe7) potentially modifies the interaction between the CNP and NEP cleavage sites, eg, at the S1 ′ subsite (For example, can be unstable). Addition of a tertiary butyl group to the smaller P2 'residue Gly8 (ie, tBu-Gly8) can potentially disrupt the interaction between CNP and the S2' subsite.

  Thus, in one aspect, CNP variants of the present disclosure have increased sensitivity to NEP cleavage by increasing the size of P1′-P2 ′ residues, eg, Phe7-Gly8, and interfering with substrate recognition at the active site. A decreasing CNP is mentioned. One or more large P1 ′ hydrophobic residues, including, but not limited to, natural amino acids, non-natural amino acids, and / or peptidomimetic moieties, including Phe7, Leu9, Leu11, Ile14, Met17, and Leu20 And / or is substituted with one or more smaller P2 ′ residues including, but not limited to, Cys6, Gly8, Gly15, Ser16, and Gly19.

The present disclosure encompasses CNP variants comprising at least one modified amino acid and / or at least one modified peptide bond, wherein at least one residue is involved in substrate recognition and / or cleavage by NEP, and the modified amino acid and modified peptide The linkage may be a natural amino acid, a non-natural amino acid, a peptidomimetic, and / or a peptide bond isostere. In one aspect, the NEP cleavage site on CNP between Cys6 and Phe7 is modified. In a related embodiment, the peptide bond between Cys6 and Phe7 (--C (= O)-NH--) is substituted with one of the following peptide bond isosteres:
--CH ₂ --NH--,
--C (= O)-N (R)-(wherein the amide group is alkylated with any of the following R groups: methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl),
_{--C (= O) - NH -} CH 2 -,
--CH ₂ --S--,
_{--CH 2 --S (O) n -} ( wherein, n is 1 or 2),
--CH ₂ --CH ₂ -,
--CH = CH--,
_{--C (= O) - CH 2} -,
--CH (CN)-NH--,
_{--CH (OH) - CH 2 -} ,
--O--C (= O) -NH--, or --NHC (= O) NH--.

によって表され、式中：
（ｘ）および（ｚ）は、独立して、不在であってもよいし、合成骨標的化合物、例えば、ビスホスホネート；関節標的または軟骨標的に有用なアミノ酸配列、例えば、ポリＡｓｐおよびポリＧｌｕ；骨タンパク質、例えば、オステオポンチン、オステオカルシン、およびシアロタンパク質の骨標的ドメインに由来するアミノ酸配列（Wang et al., Adv. Drug Delivery Rev., 57: 1049-76 (2005)）；腎クリアランスを減少させるポリマー分子および非ポリマー分子、例えば、負に帯電したＰＥＧ；ならびに、ＣＮＰ変異体の総質量を、本明細書中に一般に記載される、例えば約２．６または２．８ｋＤａから約６または７ｋＤａの範囲に増大させることによってＮＥＰ分解に対するＣＮＰ変異体の耐性を増大させる、天然のポリマー（例えば、アミノ酸、脂肪酸、および／または炭水化物を含有するもの）および合成ポリマー（例えばＰＥＧ）からなる群より選択されてもよく；
（ｂ）および（ｃ）は、野生型Ｃｙｓ６およびＰｈｅ７であっても、別の天然のアミノ酸または非天然のアミノ酸であってもよいし、ＮＥＰ切断に対する耐性を増大させるために本明細書中に記載されるペプチド結合イソスターを含有してもよく；そして
（ｄ）は、野生型Ｇｌｙ８であってもよいし、ＮＥＰへの結合を減少させるために、より大きな天然のアミノ酸もしくは非天然のアミノ酸（例えばｔ−Ｂｕ−Ｇｌｙ）、またはペプチド模倣体であってもよい。 In another embodiment, the CNP variant has the formula:

Represented by the formula:
(X) and (z) may be independently absent or a synthetic bone targeting compound, such as a bisphosphonate; an amino acid sequence useful for a joint or cartilage target, such as polyAsp and polyGlu; bone Amino acid sequences derived from bone target domains of proteins such as osteopontin, osteocalcin, and sialoprotein (Wang et al., Adv. Drug Delivery Rev., 57: 1049-76 (2005)); polymer molecules that reduce renal clearance And non-polymeric molecules, such as negatively charged PEG; and the total mass of the CNP variant is generally described herein, for example in the range of about 2.6 or 2.8 kDa to about 6 or 7 kDa. Natural polymers (eg amino acids, fatty acids) that increase the resistance of CNP variants to NEP degradation by increasing And / or those containing carbohydrates) and synthetic polymers (for example, be selected from the group consisting of PEG);
(B) and (c) can be wild-type Cys6 and Phe7, another natural or non-natural amino acid, and are used herein to increase resistance to NEP cleavage. And (d) may be wild-type Gly8 or a larger natural or non-natural amino acid (to reduce binding to NEP ( For example, it may be t-Bu-Gly), or a peptidomimetic.

  In one aspect, such CNP variants contain at least one modified amino acid at (b), (c), and / or (d).

  Other peptide bonds within CNP, including Gly8-Leu9, Lys10-Leu11, Arg13-Ile14, Ser16-Met17, and Gly19-Leu20 linkages, even if CNP22 or a variant thereof is Cys6-Phe7, are NEP resistant. Even with the peptide bond or peptide bond isoster shown, it can be cleaved. Accordingly, the present disclosure encompasses CNP variants having peptide-bound isosteres at one or more other NEP cleavage sites in addition to Cys6-Phe7 binding, and are described herein as peptide-bound isosteres. Including things.

  In another aspect, the present disclosure provides for CNP variants having cysteine analogs at Cys6 and / or Cys22, including but not limited to homocysteine, penicillamine, 2-mercaptopropionic acid, and 3-mercaptopropionic acid. Is included. In one embodiment, such a CNP variant has a cyclic domain formed by a disulfide bond between wild-type Cys6 or analog and Cys22 or analog. In yet another aspect, one or more residues, at most all of the residues of CNP22 or variants thereof are substituted with D-amino acids. Substitution of an L-amino acid with a D-amino acid essentially breaks the binding of the CNP peptide to NEP by moving the side chain about 120 degrees from its original position. In a particular embodiment, L-Phe of Phe7 is substituted with its D-enantiomer, D-Phe.

  In yet another embodiment, a beta-amino acid, such as 3-amino-2-phenylpropionic acid (or 2-phenyl-beta-alanine) replaces the wild-type alpha-amino acid Phe7. The use of beta-amino acids effectively increases the peptide backbone length by 1 methylene unit. Protease resistance can be due to changes in substrate structure or increased distance between amino acid side chains.

Non-limiting examples of variants of CNP22 having non-natural alpha-amino acids, beta-amino acids, or peptide-bound isosteres include the following:

式中：
（ｘ）および（ｚ）は、独立して、不在であってもよいし、合成骨標的化合物、例えば、ビスホスホネート；骨標的または軟骨標的に有用なアミノ酸配列、例えば、ポリＡｓｐおよびポリＧｌｕ；骨タンパク質、例えば、オステオポンチン、オステオカルシン、およびシアロタンパク質の骨標的ドメインに由来するアミノ酸配列；腎クリアランスを減少させるポリマー部分および非ポリマー部分、例えば、負に帯電したＰＥＧ；例えば、アミノ酸、疎水性の酸、および／または炭水化物を含有するポリマー；ならびに合成親水性ポリマー、例えばＰＥＧからなる群より選択されてもよく；
（ａ）は、その位置にて野生型Ｌｙｓであってもよいし、保存的アミノ酸置換基、あるいは以下に限定されないが、Ａｒｇ、Ｇｌｙ、６−ヒドロキシ−ノルロイシン、シトルリン（Ｃｉｔ）、Ｇｌｎ、Ｓｅｒ、またはＧｌｕを含む、天然もしくは非天然のアミノ酸、または側鎖上に反応性第一級アミンを有していないペプチド模倣体で置換されていてもよく、一態様において、（ａ）はＡｒｇであり；
（ｂ）は、Ｃｙｓ、およびＣｙｓ６とＰｈｅ７間のペプチド結合イソスター、例えばＣｙｓ−ＣＨ_２−−ＮＨからなる群より選択され；
（ｃ）は、Ｌ−Ｐｈｅ；Ｄ−Ｐｈｅ；３−アミノ−２−フェニルプロピオン酸；ＰｈｅのＮ−アルキル化誘導体（Ｎ−アルキル基は、メチル、エチル、ｎ−プロピル、イソプロピル、シクロプロピル、ｎ−ブチル、イソブチル、ｓｅｃ−ブチル、またはｔｅｒｔ−ブチルである）；およびＰｈｅ類似体（Ｐｈｅ類似体のベンゼン環の１つまたは複数のオルソ位置、メタ位置、および／もしくはパラ位置は、ハロゲン、ヒドロキシル、シアノ、直鎖状もしくは分枝状のＣ_１〜６アルキル、直鎖状もしくは分枝状のＣ_１〜６アルコキシ、直鎖状もしくは分枝状のハロ−Ｃ_１〜６アルキル、Ｃ_３〜１０シクロアルキル、ヘテロシクリル、Ｃ_６〜１４アリール、およびヘテロアリール（例として、以下に限定されないが、チロシン、３−クロロフェニルアラニン、２，３−クロロ−フェニルアラニン、３−クロロ−５−フルオロ−フェニルアラニン、２−クロロ−６−フルオロ−３−メチル−フェニルアラニンを含む）からなる群より選択される１つもしくは複数の置換基で置換され、または、Ｐｈｅ類似体のベンゼン環は、別のアリール基（非限定的な例として、１−および２−ナフチルアラニンを含む）で、もしくはヘテロアリール基（非限定的な例として、ピリジルアラニン、チエニルアラニン、およびフリルアラニンを含む）で置換されていてよい）からなる群より選択され；
（ｄ）は、Ｇｌｙ、ｔｅｒｔ−ブチル−Ｇｌｙ（ｔＢｕ−Ｇｌｙ）、Ｔｈｒ、Ｓｅｒ、Ｖａｌ、およびＡｓｎからなる群より選択され；
（ｅ）は、Ｌｅｕ、Ｓｅｒ、Ｔｈｒ、およびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｌｅｕからなる群より選択され；
（ｆ）は、その位置にて野生型Ｌｙｓであってもよいし、保存的アミノ酸置換基、あるいは以下に限定されないが、Ａｒｇ、Ｇｌｙ、６−ヒドロキシ−ノルロイシン、シトルリン（Ｃｉｔ）、Ｇｌｎ、Ｓｅｒ、またはＧｌｕを含む、天然もしくは非天然のアミノ酸、または側鎖上に反応性第一級アミンを有していないペプチド模倣体で置換されていてもよく、一態様において、（ｆ）はＡｒｇでなく；
（ｇ）は、Ｌｅｕおよびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｌｅｕからなる群より選択され；
（ｈ）は、Ｉｌｅ、ｔＢｕ−Ｇｌｙ、およびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｉｌｅからなる群より選択され；
（ｉ）は、Ｍｅｔ、Ｖａｌ、Ａｓｎ、ベータ−Ｃｌ−Ａｌａ、２−アミノ酪酸（Ａｂｕ）、および２−アミノ−イソ酪酸（Ａｉｂ）からなる群より選択され；そして
（ｊ）は、Ｌｅｕ、ノルロイシン（Ｎｌｅ）、ホモロイシン（Ｈｌｅｕ）、Ｖａｌ、ｔｅｒｔ−ブチル−Ａｌａ（ｔＢｕ−Ａｌａ）、Ｓｅｒ、Ｔｈｒ、Ａｒｇ、およびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｌｅｕからなる群より選択される。 In a further aspect, the CNP variant is characterized by a total mass generally as described herein, eg, ranging from about 2.6 or 2.8 kDa to about 6 or 7 kDa, and resistant to NEP degradation. Designed for augmentation and represented by the following formula:

In the formula:
(X) and (z) may be independently absent or a synthetic bone targeting compound, such as a bisphosphonate; an amino acid sequence useful for bone or cartilage targeting, such as polyAsp and polyGlu; bone Amino acid sequences derived from bone target domains of proteins such as osteopontin, osteocalcin, and sialoprotein; polymeric and non-polymeric moieties that reduce renal clearance, such as negatively charged PEG; such as amino acids, hydrophobic acids, And / or a polymer containing carbohydrates; and may be selected from the group consisting of synthetic hydrophilic polymers such as PEG;
(A) may be wild-type Lys at that position, conservative amino acid substituent, or, but not limited to, Arg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln, Ser Or a natural or non-natural amino acid, including Glu, or a peptidomimetic that does not have a reactive primary amine on the side chain, and in one embodiment (a) is Arg Yes;
(B) is, Cys, and Cys6 and Phe7 between the peptide bond isostere, for example selected from the group consisting of _Cys-CH 2 --NH;
(C) is L-Phe; D-Phe; 3-amino-2-phenylpropionic acid; N-alkylated derivative of Phe (N-alkyl groups are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl); and a Phe analog (one or more ortho, meta, and / or para positions of the benzene ring of the Phe analog are halogen, Hydroxyl, cyano, linear or branched C _1-6 alkyl, linear or branched C _1-6 alkoxy, linear or branched halo-C _1-6 alkyl, C _{3 10} cycloalkyl, _{heterocyclyl, C having 6 to 14} aryl, and a heteroaryl (e.g., but not limited to, tyrosine, 3- chlorophyll One or more substituents selected from the group consisting of nylalanine, 2,3-chloro-phenylalanine, 3-chloro-5-fluoro-phenylalanine, 2-chloro-6-fluoro-3-methyl-phenylalanine Or the benzene ring of the Phe analog is replaced with another aryl group (including, as non-limiting examples, 1- and 2-naphthylalanine) or a heteroaryl group (as non-limiting example, Selected from the group consisting of (optionally) substituted with pyridylalanine, thienylalanine, and furylalanine;
(D) is selected from the group consisting of Gly, tert-butyl-Gly (tBu-Gly), Thr, Ser, Val, and Asn;
(E) is selected from the group consisting of Leu, Ser, Thr, and peptide-bound isosteres such as N-Me-Leu;
(F) may be wild-type Lys at that position, conservative amino acid substituent, or, but not limited to, Arg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln, Ser Or a natural or non-natural amino acid, including Glu, or a peptidomimetic that does not have a reactive primary amine on the side chain, and in one embodiment (f) is Arg None;
(G) is selected from the group consisting of Leu and peptide bond isosteres such as N-Me-Leu;
(H) is selected from the group consisting of Ile, tBu-Gly, and peptide-bound isosteres such as N-Me-Ile;
(I) is selected from the group consisting of Met, Val, Asn, Beta-Cl-Ala, 2-aminobutyric acid (Abu), and 2-amino-isobutyric acid (Aib); and (j) is Leu, Norleucine (Nle), homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Ser, Thr, Arg, and peptide-bound isosteres such as N-Me-Leu.

式中：
（ｘ）および（ｚ）は、独立して、不在であってもよいし、合成骨標的化合物、例えばビスホスホネート；骨標的または軟骨標的に有用なアミノ酸配列、例えば、ポリＡｓｐおよびポリＧｌｕ；骨タンパク質およびその誘導体の骨標的ドメイン、例えば、オステオポンチン、オステオカルシン、シアロタンパク質等の融合タンパク質またはペプチド配列に由来するアミノ酸配列；以下に限定されないが、親水性ポリマーまたは水溶性ポリマー、例えば、帯電したＰＥＧ分子を含む、腎クリアランスを減少させる部分；ならびに、例えば、ＰＥＧ、アミノ酸、炭水化物、および／または疎水性の酸を含む部分からなる群より選択されてもよく；
（ａ）は、その位置にて野生型Ｌｙｓであってもよいし、保存的アミノ酸置換基、あるいは以下に限定されないが、Ａｒｇ、Ｇｌｙ、６−ヒドロキシ−ノルロイシン、シトルリン（Ｃｉｔ）、Ｇｌｎ、Ｓｅｒ、またはＧｌｕを含む、天然もしくは非天然のアミノ酸、または側鎖上に反応性第一級アミンを有していないペプチド模倣体で置換されていてもよく、一態様において、（ａ）はＡｒｇであり；
（ｂ）は、Ｃｙｓ、およびＣｙｓ６とＰｈｅ７間のペプチド結合イソスター、例えばＣｙｓ−ＣＨ_２−−ＮＨからなる群より選択され；
（ｃ）は、Ｌ−Ｐｈｅ；Ｄ−Ｐｈｅ；３−アミノ−２−フェニルプロピオン酸；ＰｈｅのＮ−アルキル化誘導体（Ｎ−アルキル基は、メチル、エチル、ｎ−プロピル、イソプロピル、シクロプロピル、ｎ−ブチル、イソブチル、ｓｅｃ−ブチル、またはｔｅｒｔ−ブチルである）；およびＰｈｅ類似体（Ｐｈｅ類似体のベンゼン環の１つまたは複数のオルソ位置、メタ位置、および／もしくはパラ位置は、ハロゲン、ヒドロキシル、シアノ、直鎖状もしくは分枝状のＣ_１〜６アルキル、直鎖状もしくは分枝状のＣ_１〜６アルコキシ、直鎖状もしくは分枝状のハロ−Ｃ_１〜６アルキル、Ｃ_３〜１０シクロアルキル、Ｃ_６〜１４アリール、ヘテロシクリル、およびヘテロアリール（例として、以下に限定されないが、チロシン、３−クロロフェニルアラニン、２，３−クロロ−フェニルアラニン、３−クロロ−５−フルオロ−フェニルアラニン、２−クロロ−６−フルオロ−３−メチル−フェニルアラニンを含む）からなる群より選択される１つもしくは複数の置換基で置換され、または、Ｐｈｅ類似体のベンゼン環は、別のアリール基（非限定的な例として、１−および２−ナフチルアラニンを含む）で、もしくはヘテロアリール基（非限定的な例として、ピリジルアラニン、チエニルアラニン、およびフリルアラニンを含む）で置換されていてよい）からなる群より選択され；
（ｄ）は、Ｇｌｙ、ｔｅｒｔ−ブチル−Ｇｌｙ、Ｔｈｒ、Ｓｅｒ、Ｖａｌ、およびＡｓｎからなる群より選択され；
（ｅ）は、Ｌｅｕ、Ｓｅｒ、Ｔｈｒ、およびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｌｅｕからなる群より選択され；
（ｆ）は、Ｌｙｓ、Ａｒｇ、Ｇｌｙ、６−ヒドロキシ−ノルロイシン、シトルリン（Ｃｉｔ）、Ｇｌｎ、およびＳｅｒからなる群より選択され；
（ｇ）は、Ｌｅｕ、Ａｓｎおよびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｌｅｕからなる群より選択され；
（ｈ）は、Ｉｌｅ、ｔｅｒｔ−ブチル−Ｇｌｙ（ｔＢｕ−Ｇｌｙ）、Ａｓｎ、およびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｉｌｅからなる群より選択され；
（ｉ）は、Ｇｌｙ、Ａｒｇ、Ｓｅｒ、およびＡｓｎからなる群より選択され；
（ｊ）は、Ｍｅｔ、Ｖａｌ、Ａｓｎ、ベータ−Ｃｌ−Ａｌａ、２−アミノ酪酸（Ａｂｕ）、および２−アミノ−イソ酪酸（Ａｉｂ）からなる群より選択され；そして
（ｋ）は、Ｌｅｕ、ノルロイシン（Ｎｌｅ）、ホモロイシン（Ｈｌｅｕ）、Ｖａｌ、ｔｅｒｔ−ブチル−Ａｌａ（ｔＢｕ−Ａｌａ）、Ａｒｇ、Ｔｈｒ、Ｓｅｒ、およびペプチド結合イソスター、例えばＮ−Ｍｅ−Ｌｅｕからなる群より選択される。 In another embodiment, the CNP variant is characterized by a total mass that is generally described herein, eg, ranging from about 2.6 or 2.8 kDa to about 6 or 7 kDa, and resistant to NEP cleavage. Designed for augmentation and represented by the following formula:

In the formula:
(X) and (z) may be independently absent or a synthetic bone targeting compound, such as a bisphosphonate; an amino acid sequence useful for bone or cartilage targeting, such as polyAsp and polyGlu; bone protein And amino acid sequences derived from bone targeting domains of derivatives thereof, such as fusion proteins or peptide sequences such as osteopontin, osteocalcin, sialoprotein, etc .; but not limited to hydrophilic polymers or water-soluble polymers such as charged PEG molecules A moiety that reduces renal clearance; and may be selected from the group consisting of, for example, moieties comprising, for example, PEG, amino acids, carbohydrates, and / or hydrophobic acids;
(A) may be wild-type Lys at that position, conservative amino acid substituent, or, but not limited to, Arg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln, Ser Or a natural or non-natural amino acid, including Glu, or a peptidomimetic that does not have a reactive primary amine on the side chain, and in one embodiment (a) is Arg Yes;
(B) is, Cys, and Cys6 and Phe7 between the peptide bond isostere, for example selected from the group consisting of _Cys-CH 2 --NH;
(C) is L-Phe; D-Phe; 3-amino-2-phenylpropionic acid; N-alkylated derivative of Phe (N-alkyl groups are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl); and a Phe analog (one or more ortho, meta, and / or para positions of the benzene ring of the Phe analog are halogen, Hydroxyl, cyano, linear or branched C _1-6 alkyl, linear or branched C _1-6 alkoxy, linear or branched halo-C _1-6 alkyl, C _{3 10 cycloalkyl, C having 6 to 14} aryl, heterocyclyl, and as heteroaryl (e.g., but not limited to, tyrosine, 3- chlorophyll One or more substituents selected from the group consisting of nylalanine, 2,3-chloro-phenylalanine, 3-chloro-5-fluoro-phenylalanine, 2-chloro-6-fluoro-3-methyl-phenylalanine Or the benzene ring of the Phe analog is replaced with another aryl group (including, as non-limiting examples, 1- and 2-naphthylalanine) or a heteroaryl group (as non-limiting example, Selected from the group consisting of (optionally) substituted with pyridylalanine, thienylalanine, and furylalanine;
(D) is selected from the group consisting of Gly, tert-butyl-Gly, Thr, Ser, Val, and Asn;
(E) is selected from the group consisting of Leu, Ser, Thr, and peptide-bound isosteres such as N-Me-Leu;
(F) is selected from the group consisting of Lys, Arg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln, and Ser;
(G) is selected from the group consisting of Leu, Asn and peptide-bound isosteres such as N-Me-Leu;
(H) is selected from the group consisting of Ile, tert-butyl-Gly (tBu-Gly), Asn, and peptide-bound isosteres, such as N-Me-Ile;
(I) is selected from the group consisting of Gly, Arg, Ser, and Asn;
(J) is selected from the group consisting of Met, Val, Asn, Beta-Cl-Ala, 2-aminobutyric acid (Abu), and 2-amino-isobutyric acid (Aib); and (k) is Leu, It is selected from the group consisting of norleucine (Nle), homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Arg, Thr, Ser, and peptide-bound isosteres such as N-Me-Leu.

  In order to improve delivery of CNP variants to target sites of joint-related disorders (eg osteoarthritis), CNP variants may be bone target or cartilage target moieties (eg, at the N-terminus and / or C-terminus). May be attached to. Non-limiting examples of bone or cartilage target portions include bisphosphonates; hydroxyapatite; glucosamine; collagen (eg, type X collagen); poly Asp; poly Glu; and bone proteins such as osteocrine, osteopontin, osteocalcin, and Examples include amino acid sequences derived from the bone target domain of sialoprotein.

  In addition to being less sensitive to NEP cleavage, CNP variants potentially retain CNP functionality while having low affinity for the NPR-C clearance receptor. In addition to NEP-mediated degradation, the half-life of CNP22 is affected by the clearance receptor, NPR-C, which shares 58% sequence homology with the extracellular peptide binding domain of NPR-B. CNP22 binds not only to NPR-B (7-30 pM affinity) but also to NPR-C (11-140 pM) (Bennett, BD et al., J. Biol. Chem., 266). : 23060-67 (1991); Koller, KJ & Goeddel, DV, Circulation, 86: 1081-88 (1992); Suga, S. et al., Endocrinology, 130: 229-39 (1992)). The NPR-B crystal structure has not yet been reported, but sequence homology and similarity between the crystal structures of NPR-C and NPR-A (He, X.-L. et al., Science, 293 (5535): 1657-62 (2001); Ogawa, H. et al., J. Biol. Chem., 279 (27): 28625-31 (2004); He, X.-L., J. Mol. Biol., 361 (4): 698-714 (2006)) suggests that NPR-B is probably considered to be similar in overall structural folding.

  Therefore, an NPR-B homology model was constructed based on the structure-based sequence alignment and crystal structure of the following related systems: CNP bound to NPR-C, ANP bound to NPR-A, and NPR-C. Bound ANP (He, X.-L. et al., Science, 293 (5535): 1657-62 (2001); Ogawa, H. et al., J. Biol. Chem., 279 (27): 28625 -31 (2004); He, X.-L., J. Mol. Biol., 361 (4): 698-714 (2006)). Based on the observation that the receptor is likely to determine the bound peptide structure and NPR-B is closest to NPR-A with respect to primary structure and functional properties, the NPR-B / CNP homology model is modeled NPR-A / ANP crystal structure. Published signaling data for CNP variants (US Patent No. 5,434,133 and US Patent Application Publication No. 2004 / 0138134A1), and published functional ANP variants that no longer bind to NPR-C The NPR-B / CNP model was refined using existing signaling data (Cunningham, EMBO 13 (11) 2508-15, 1994).

  The present disclosure encompasses CNP variants designed for improved NPR-B selectivity based on homology-based structural models of the NPR-B / CNP complex. Combining experimental computer structure data for natriuretic peptides bound to various receptors with published functional data continues to bind to NPR-B, but increases affinity for NPR-C clearance receptors. CNP variants have been generated that can be potentially reduced. For example, NPR-C has a unique insertion within the loop structure at the peptide binding site, and its loop residues are compared to the respective loop residues in NPR-A and NPR-B, and CNP Gly8 (Or ANP Gly9) is located closer to the peptide residue. Previous studies have shown that G9T mutations in ANP contribute to improving NPR-A selectivity by reducing affinity for NPR-C (Cunningham, EMBO J., 13 (11): 2508-15 (1994)). Thus, replacing the corresponding Gly8 residue with a larger residue (Ser, Val, Thr, or Asn) breaks CNP binding to NPR-C without affecting binding to NPR-B CNP mutants were created. In addition, one or more mutations were introduced at the C-terminus of CNP, including Gly15 to Gly21. This is expected to interact with receptor-specific residues based on detailed structural analysis of the receptor / peptide complex. For example, the G19R mutation in CNP22 does not significantly lose NPR-B signaling activity. However, this mutation cannot be modeled into an effective crystal structure of NPR-C / CNP without altering the structure of adjacent residues. These observations suggest that G19R mutations can selectively disrupt CNP binding to certain receptors, such as NPR-C.

  In one aspect, the CNP variant has a substitution at one or more Gly sites at positions 1, 5, 8, 15, 19, and 21 to reduce the conformational freedom. Increases body specificity. Comparative analysis of the crystal structure of ANP bound to NPR-C and NPR-A (Ogawa, H. et al., J. Biol. Chem., 279: 28625-31 (2004); He, X.-L., J. Mol. Biol., 361: 698-714 (2006)) shows that the conformational freedom of ANP can play an important role in determining receptor selectivity.

  In one aspect, a CNP functional variant with potentially reduced affinity for NPR-C has one or more of the following amino acid substitutions: G1R, G1E, GSR, G5Q, G5S, F7Y, G8T, G8S, G8V, G8N, L9S, L9T, K10Cit, K10Q, K10S, I14N, G15R, G15S, G15N, G15Cit, S16Q, M17V, M17N, G19S, G19R, G19N, L20V, L20R, L20T, L20S, G21S And G21R. In one embodiment, the CNP variant has a multipoint substitution at positions 1, 5, 7, 8, 9, 10, 14, 15, 16, 17, 19, 20, and / or 21, and optionally , May have modifications at any of the other positions in the peptide sequence of the variant.

  In further embodiments, the CNP variants described herein are conjugated to a moiety and the maximum total mass is generally as defined herein at the N-terminus, C-terminus, and / or internal site. Described, eg, characterized by a range of about 2.6 or 2.8 kDa to about 6 or 7 kDa, promotes joint / cartilage targeting, reduces NPR-C and renal clearance, and increases resistance to NEP degradation And / or improve CNP functionality. In one aspect, the CNP variant is not bound to a polymer moiety at a site within the cyclic region (corresponding to Cys6 to Cys22 of CNP22). Non-limiting examples of polymeric or non-polymeric moieties that can be conjugated to CNP variants include synthetic bone targeting compounds such as bisphosphonates; bone / cartilage targeting peptide sequences such as polyAsp and polyGlu; bone proteins such as osteopontin, osteocalcin And peptide sequences derived from bone targeting domains of sialoproteins; peptide sequences derived from functional domains of bone morphogenetic proteins such as BMP2, BMP3, BMP5, BMP7, and BMP8a; natriuretic sources such as NPPC, ANP, and Peptide sequences derived from BNP polypeptides; other natural or non-polymeric moieties such as carbohydrates, fatty acids, and phospholipids; biocompatible synthetic hydrophilic polymers such as PEG (or PEO); Sex polymer moiety or a non-polymeric moiety, for example, heptanoic acid and pentanoic acid; and combinations thereof.

  The CNP variants described herein may be substantially similar in functional activity to CNP22 or better than CNP22, eg, with respect to cGMP production and signaling stimulation. In one aspect, the CNP variant stimulates in vitro or in vivo production of at least about 50% of cGMP levels produced under the same concentration of wtCNP22 (eg, 1 μM). In certain embodiments, the CNP variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the in vitro or in vivo cGMP stimulating activity of wild-type CNP22. In another embodiment, the CNP variant has improved cGMP stimulating activity as compared to CNP22. In certain embodiments, the CNP variant is at least about 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300 of cGMP levels produced under the same concentration of wtCNP22 (eg, 1 μM). %, 350%, 400%, 450%, 500%, or more production is stimulated in vitro or in vivo.

C. Synthesis and Purification of CNP Variants In some embodiments, CNP variants useful herein are generated by recombinant expression in certain embodiments using certain techniques known in the art. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY (1989)); DNA Cloning: A Practical Approach, Volumes I and II, DN Glover, Ed (1985); and Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

  In certain embodiments, the CNP variant comprises a host cell comprising a first polynucleotide encoding a CNP variant polypeptide linked in a medium to a second polynucleotide encoding a cleavable peptide or protein. Produced by a recombinant process comprising culturing under conditions that result in expression of a fusion polypeptide encoded by the polynucleotide, wherein the fusion polypeptide is directly linked to a cleavable peptide or protein, or a linker A CNP variant polypeptide indirectly linked via In some embodiments, the host cell has been transformed with an expression vector comprising a polynucleotide encoding a CNP variant polypeptide linked to a polynucleotide encoding a cleavable peptide or protein. In certain embodiments, the fusion polypeptide is expressed as a soluble protein or as an inclusion. The expressed fusion polypeptide can be isolated from the host cell or culture medium, and the isolated fusion polypeptide can be contacted with a cleaving agent to release the CNP variant.

  Methods for generating CNP variants are described in US Pat. No. 8,198,242, US Pat. No. 8,377,884, and US Pat. No. 8,598,121, incorporated herein by reference. Have been described.

D. Chemically modified CNP variants Chemical modification of CNP variants can potentially lead to modified CNP peptides with advantageous properties such as increased stability and half-life, and decreased immunogenicity ( For general considerations of chemical modification of therapeutic proteins, see Pharmazie, 57 (1): 5-29 (2002)). In addition to pegylation, glycosylation, and other chemical derivatization procedures, for example, phosphorylation, amidation, carboxylation, acetylation, modification by methylation, and acid addition salts, amides, esters, and N-acyls Derivative generation can also potentially mask immunogenic and / or proteolytic sensitive areas (Science, 303: 480-482 (2004)).

  Examples of chemical modifications include, but are not limited to, Bednarsaki's polymer addition method and Altus Corporation's cross-linking method to improve stability and protease resistance and reduce immunogenicity. Bednarsaki shows that polymer addition can improve the temperature stability of proteins (J. Am. Chem. Soc., 114 (1): 378-380 (1992)), Altus Corporation has found that glutaraldehyde crosslinking is an enzyme. It has been found that stability can be improved.

  Chemical modification of the polypeptide may be performed non-specifically (resulting in a mixture of derivatized species) or site-specific (eg, site-directed mutagenesis). And based on wild-type macromolecular reactivity-directed derivatization and / or site-selective modification using a combination of chemical modifications), instead, ligation methods for expressed proteins (Curr. Opin. Biotechnol. 13 (4): 297-303 (2002)).

PEGylated CNP Variants In one embodiment, CNP variants (including those with amino acid additions, substitutions, and / or deletions) bind to hydrophilic natural or synthetic polymers for increased stability. Is done. In one aspect, since the hydrophilic polymer is water soluble, the CNP peptide bound thereto does not precipitate in an aqueous (eg, physiological) environment. Furthermore, hydrophilic polymers are biocompatible, i.e. do not cause damage, toxicity or immunological reactions in vivo. The hydrophilic polymer may be branched or unbranched. In one embodiment, the hydrophilic polymer is not branched.

  Various binding sites of the CNP variant to the hydrophilic polymer are contemplated, including but not limited to: (1) N-terminal only; (2) C-terminal only; (3) Internal sites only (eg, Lys4); (4) both N-terminal and C-terminal; (5) N-terminal and internal sites; and (6) C-terminal and internal sites. In one embodiment, the CNP variant is attached to the hydrophilic polymer only at the N-terminus. In another embodiment, the binding is only at an internal site (eg, Lys4). In yet another embodiment, the linkage is made at the N-terminus and at an internal site (eg, Lys4). In yet another aspect, for better functionality, CNP variants are not attached to hydrophilic polymers at sites within the cyclic domain (corresponding to Cys6 to Cys22 of CNP-22) (eg, Lys10). . If binding to the hydrophilic polymer is based on bond formation with a reactive primary amino group on the CNP variant, binding at an internal site (eg, Lys4 and / or Lys10) will result in Lys4 and / or Lys10 By substitution with a natural or non-natural amino acid or peptidomimetic that does not contain a reactive primary amino group on the side chain, eg, Gly, Ser, Arg, Asn, Gln, Asp, Glu, or citrulline (Cit) Can be hindered. In certain embodiments, Lys4 and / or Lys10 is substituted with Arg. In another embodiment, Lys10 is not substituted with Arg.

Non-limiting examples of hydrophilic polymers include polymers formed from monomers having carboxylic acids (eg, methacrylic acid (MA) and acrylic acid (AA)), polyvinyl alcohol, monomers having hydroxyl groups (eg, hydroxyethyl Polymers formed from methacrylate (HEMA), hydroxypropyl methacrylate (HPMA), hydroxypropyl methacrylamide, and 3-trimethylsilylpropyl methacrylate (TMSPMA)), polyalkylene oxides, polyoxyethylated polyols (eg glycerol), poly (ethylene glycol) (PEG), poly (propylene glycol), mono -C1～C ₁₀ alkoxy-PEG (e.g. monomethoxy-PEG), tresyl monomethoxy-PEG, Ali Oxy-PEG, PEG acrylate (PEGA), PEG methacrylate, PEG propionaldehyde, bis-succinimidyl carbonate PEG, copolymer of 2-methacryloyloxyethyl-phosphorylcholine (MPC) and N-vinylpyrrolidone (VP), hydroxy-functional poly (N-vinylpyrrolidone) (PVP), SIS-PEG (SIS is a polystyrene-polyisobutylene-polystyrene block copolymer), polystyrene-PEG, polyisobutylene-PEG, PCL-PEG (PCL is polycaprolactone) , PLA-PEG (PLA is polylactic acid), PMMA-PEG (PMMA is poly (methyl methacrylate)), PDMS-PEG (PDMS is polydimethyloxanone) ), PVDF-PEG (PVDF is polyvinylidene fluoride), PLURONIC ™ surfactant (polypropylene oxide-co-polyethylene glycol), poly (tetramethylene glycol), poly (L-lysine-g-ethylene) Glycol) (PLL-g-PEG), poly (L-lysine-g-hyaluronic acid) (PLL-g-HA), poly (L-lysine-g-phosphorylcholine) (PLL-g-PC), poly (L -Lysine-g-vinylpyrrolidone) (PLL-g-PVP), poly (ethylimine-g-ethylene glycol) (PEI-g-PEG), poly (ethylimine-g-hyaluronic acid) (PEI-g-HA), Poly (ethylimine-g-phosphorylcholine) (PEI-g-PC), poly (ethylimine-g-vinylpyrrolidone) (PEI-g-PVP), PLL-co-HA, PLL-co-PC, PLL-co-PVP, PEI-co-PEG, PEI-co-HA, PEI-co-PC, PEI-co-PVP, Cellulose and its derivatives (for example, hydroxyethyl cellulose), dextran, dextrin, hyaluronic acid and its derivatives (for example, sodium hyaluronate), elastin, chitosan, acrylic sulfate, acrylic sulfonate, acrylic sulfamate, methacrylic sulfate, methacrylic sulfonate, methacrylic sulfa Mate, polymers and copolymers thereof, and polymers and copolymers of combinations thereof.

In certain embodiments, the hydrophilic polymer is poly (ethylene glycol) (PEG), also referred to as poly (ethylene oxide) (PEO). The term “PEG” or “PEO” as used herein includes all branched and unbranched forms of PEG, including but not limited to mono (C ₁ -C ₁₀ ) alkoxy. Can be used to derivatize polypeptides including -PEG and aryloxy-PEG.

In one embodiment, PEG-CNP-linked comprises PEG (or PEO) polymer of the formula _{(CH 2 CH 2 O) n} , wherein, n is an integer from about 6 to about 100, the PEG polymer, About 0.3 kDa to about 5 kDa. In another embodiment, n is an integer from about 12 to about 50 and the PEG polymer is from about 0.6 kDa to about 2.5 kDa. In yet another aspect, n is from about 12 to about 24 and the PEG polymer is from about 0.6 kDa to about 1.2 kDa. In further embodiments, the terminal hydroxyl groups of the PEG polymer are capped with non-reactive groups. In certain embodiments, the end capping group is an alkyl group, eg, a lower alkyl group such as methyl, so the PEG polymer ends with an alkoxy group. In one embodiment, the PEG polymer is not branched. In another embodiment, CNP-22 or a variant thereof is attached to the PEG polymer only at the N-terminus.

PEG and PEO potentially include molecules with a distribution of molecular weight, ie molecules that are potentially polydispersed, depending on the method being prepared. The size / mass distribution of the polymer preparation can be statistically characterized by its weight average molecular weight (M _w ) and its number average molecular weight (M _n ), the ratio of which is the polydispersity index (M _w / M _n ) and be called. M _w and M _n can be measured by mass spectroscopy. PEG-CNP variants attached to PEG moieties greater than 1.5 kDa may exhibit a wide range of molecular weights due to the polydispersity of the parent PEG molecule. For example, in the case of mPEG2K (Sunbright ME-020HS, NOF Co.), the molecular mass of PEG molecules is distributed over a range of about 1.5 kDa to about 3 kDa, and the polydispersity index is 1.036. On the other hand, using Pierce Biotechnology (Rockford, Illinois) MS (PEG) _n reagent (n = 4, 8, 12, or 24, eg expressed as “PEO12” or “PEO24”), CNP− PEG attached to 22 or a variant thereof is monodisperse, the chain length is discontinuous, and the molecular weight is defined.

Methods for producing polypeptides containing PEG moieties are known in the art (see, eg, US Pat. No. 5,824,784). Methods for preparing PEGylated CNP peptides generally involve (a) reacting CNP-22 or a variant thereof with a PEGylation reagent under conditions suitable for attaching PEG to the CNP peptide (eg, at the N-terminus). And (b) obtaining a reaction product. By PEGylating the CNP peptide, the binding to NPR-B can vary greatly depending on the size of the PEG moiety and the location of the PEGylation, so various types of PEG and PEGylation reaction conditions can be explored. As a chemical action that can be used for pegylation of CNP peptide, the use of NHS-ester of methoxy PEG (O-[(N-succinimidyloxycarbonyl) -methyl] -O'-methylpolyethyleneglycol) Examples include acylation of reactive primary amines. Acylation with methoxy-PEG-NHS or methoxy-PEG-SPA results in an amide bond, eliminating any charge of the original primary amine. PEG-CNP variants designated by the designation “PEO12” or “PEO24”, as well as PEG-CNP variants designated by the designation “PEG1K”, “PEG2K”, “PEG5K” or “PEG20K” Pegylated via reaction of primary amino group with NHS ester activated methoxy end-capped PEG reagent. PEG-CNP Variants also by other methods, for example, primary amino group and a PEG aldehyde of the peptide, for example, PEG-propionaldehyde or mono _-C 1 _{-C 10} alkoxy or aryloxy derivatives thereof, involving reduction It may be prepared via amination (see US Pat. No. 5,252,714).

  Unlike ribosomal protein synthesis, synthetic peptide synthesis proceeds from the C-terminus to the N-terminus. Thus, Boc-PEG (containing tert-butyloxycarbonyl (Boc)) is one way of attaching PEG to the C-terminus of the peptide (RB Merrifield, J. Am. Chem. Soc., 85 (14): 2149-2154 (1963)). In addition, Fmoc (fluorenylmethoxycarbonyl) chemicals can be used (E. Atherton and RC Sheppard, Solid Phase Peptide Synthesis: a Practical Approach, IRL Press (Oxford, England (1989)).

  The method of preparing PEG-CNP variants provides a substantially homogeneous mixture of polymer-protein bound forms. The discontinuous PEG-CNP preparation after purification is sufficiently pure for in vitro and in vivo testing of biological properties. The nature and extent of pegylation can be determined using, for example, PAGE and HPLC analysis. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the CNP variants are monopegylated at the N-terminus. In order to optimize the beneficial effect of PEGylation on the biological properties of CNP, the polymer length, structure (eg, branched or linear) and / or functionalized (eg, negatively charged) of the PEG moiety Group addition) may be varied. Pegylated CNP variants are tested for NEP resistance, pharmacokinetics, and biological activity (eg, the ability to bind to NPR-B and stimulate cGMP production). PEGylated CNP mutants that exhibit improved NEP resistance and at least about 50% of CNP-22's cGMP-stimulating activity are further expressed in, for example, rat chondrosarcoma cell-based chondrogenic models in vitro, and mouse chondrogenic animals It can be tested in vivo in a model.

E. Methods Using CNP Variants, Pharmaceutical Compositions of CNP Variants, and Methods Using Routes of Administration CNP Variants As described in the Examples below, the experiments reported here are disclosed herein. Provides evidence that the CNP variants produced are useful in the treatment of osteoarthritis. Accordingly, aspects of the invention include administering a CNP variant to a subject diagnosed with osteoarthritis. As a result, the rate of cartilage degeneration in a subject can be reduced and in some cases reversed (ie, cartilage can be induced to regenerate). Thus, various symptoms and biomarkers associated with osteoarthritis can be improved. The examples show the utility of Gly-CNP-37 and Pro-Gly-CNP-37, but other variants disclosed herein can also be tested for efficacy and used in the same way . In particular, Gly-CNP-37 and Pro-Gly-CNP-37 show a dramatic improvement in cartilage growth, motility, and motility techniques, and a signi fi cant decrease in cartilage degeneration.

  Osteoarthritis can be diagnosed based on reports of signs and symptoms from patients, as well as based on medical images to determine cartilage damage in a particular joint, as described above. Imaging methods include radiography, magnetic resonance imaging (MRI), optical coherence tomography (OCT), and ultrasound (US) (Gold et al., Bone J. 51: 278-88). , 2012). Imaging can often detect joint narrowing, bone growth and subchondral cysts in the joint area, articular cartilage thinning, degenerative subchondral bone marrow changes, and bone fusion. In various embodiments, administration of a CNP variant contemplated herein may result in cartilage degeneration, cartilage growth, patient motility, joint narrowing, bone growth, and subchondral cyst, articular cartilage thinning in the joint area. Improves one or more symptoms of osteoarthritis described herein, including degenerative subchondral bone marrow changes, as well as bone fusion, as well as others known in the art.

  In certain instances, osteoarthritis was divided into primary osteoarthritis and secondary osteoarthritis (Stacy et al., Primary Osteoarthritis Imaging, 2012). Primary arthritis refers to an idiopathic phenomenon that often occurs in older individuals and in previously intact joints without apparent onset factors. Secondary osteoarthritis can refer to a degenerative disease of the synovial joint due to some predisposing condition, usually trauma that adversely affects the articular cartilage and / or subchondral bone of the affected joint. Secondary osteoarthritis can occur in relatively young individuals. Treatment of both primary and secondary osteoarthritis is contemplated herein.

  The severity of arthritis is determined by Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) (Kirkley et al., N Engl J Med 359: 1097-1107, 2008), Short Form-36 (SF-36) Physical Component Summary (Ware et al., Med Care 30: 473-483, 1992), and Arthritis Self-Efficacy Scale (ASES) (Lorig et al., Arthritis Rheum 32: 37-44, 1989). The severity of radiography is assessed by a modified Kellgren-Lawrence classification (Kellgren et al., Ann Rheum Dis 1957; 16: 494-502; Blackburn et al., J Rheumatol; 21: 675-679, 1994) Can be done.

  Osteoarthritis can affect cartilage in joints including, but not limited to, knees, shoulders, elbows, fingers, hands, wrists, hips, ankles, neck, spine, and / or lower back. If the arthritis is of the back, symptoms of osteoarthritis include joint pain, joint stiffness, joint swelling, decreased range of motion, and weakness or paralysis of the arms and legs. There is currently an unmet need for new effective treatments for osteoarthritis. By promoting chondrocyte matrix production and growth and differentiation, the CNP variants described herein are subject to arthritis, including osteoarthritis, and to counter the undesirable effects of FGF-2 Are useful for treating arthritis, including osteoarthritis, by increasing matrix synthesis.

  In various embodiments, cartilage damage or growth is analyzed by chondrocyte death / loss, proteoglycan (PG) loss, and collagen loss or fibrillation. Cartilage damage and improvement after treatment can also be assessed by measuring the level of at least one cartilage-specific biomarker described herein. The improvement in exercise capacity may be monitored by any exercise capacity and rating scale described herein and known in the art.

  Treatment of osteoarthritis often includes pain management with anti-inflammatory drugs such as NSAIDS including aspirin, naproxen or ibuprofen, acetaminophen, glucocorticoids, steroids, and hyaluronic acid. The moderate to severe form of arthritis can also be treated by surgery to remove some cartilage that can replace the affected joint or cause pain (Kirkey et al., N Engl J Med 359: 1097). -1107, 2008). The CNP variants herein may be administered in conjunction with another procedure commonly used to treat osteoarthritis as described herein or known to the treating physician.

Pharmaceutical compositions of CNP variants In an additional aspect, the present disclosure provides pharmaceutical compositions comprising a CNP variant and one or more pharmaceutically acceptable excipients, carriers and / or diluents. Intended for use. In certain embodiments, the composition further comprises one or more other biologically active agents (eg, protease, receptor tyrosine kinase, and / or inhibitor of clearance receptor NPR-C).

  In some embodiments, the composition comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the desired CNP variant. Contains by purity. In certain embodiments, the composition comprises about 10%, 5%, 4%, 3%, 2%, 1% of polymeric contaminants such as other mammalian (eg human) proteins and other CNP variants. Or it contains less than 0.5%.

Non-limiting examples of excipients, carriers, and diluents include vehicles, liquids, buffers, isotonic agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, An adjuvant etc. are mentioned. The composition is a liquid (eg, water, ethanol); various buffer contents (eg, Tris-HCl, phosphate, acetate buffer, citrate buffer), pH and ionic strength diluents; detergents and solubilizers ( For example, Polysorbate 20, Polysorbate 80); antioxidants (eg, methionine, ascorbic acid, sodium metabisulfite); preservatives (eg, Thimerosol, benzyl alcohol, m-cresol); and bulking substances (eg, lactose, mannitol) Sucrose). Excipient in the formulation of the pharmaceutical composition, the use of diluents and carriers are known in the art; for ^{example, Remington's Pharmaceutical Sciences, 18 th} Edition, pages 1435-1712, Mack Publishing Co. (Easton, See Pennsylvania (1990)). This document is incorporated herein by reference in its entirety.

  For example, carriers include, but are not limited to, diluents, vehicles and adjuvants that do not react with the active ingredients, as well as implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials. Non-limiting examples of carriers include phosphate buffered saline, saline, water, and emulsions (eg oil / water emulsions). The carrier can be a solvent or dispersion medium containing, for example, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, and mixtures thereof.

  In some embodiments, the composition is a liquid formulation. In certain embodiments, the formulation comprises CNP variant from about 0.1 mg / ml to about 20 mg / ml, from about 0.5 mg / ml to about 20 mg / ml, from about 1 mg / ml to about 20 mg / ml, about 0.1 mg / ml. In a concentration range of 1 mg / ml to about 10 mg / ml, about 0.5 mg / ml to about 10 mg / ml, or about 1 mg / ml to about 10 mg / ml.

  In a further embodiment, the composition comprises a buffer solution or buffering agent for maintaining the pH of the CNP-containing solution or suspension containing the desired range. Non-limiting examples of buffer solutions include phosphate buffered saline, Tris buffered saline, and Hank buffered saline. Buffering agents include but are not limited to sodium acetate, sodium phosphate, and sodium citrate. Mixtures of buffering agents may be used. In certain embodiments, the buffering agent is acetic acid / acetate or citric acid / citrate. The amount of a suitable buffer in the composition will depend, in part, on the particular buffer used and the desired pH of the solution or suspension. For example, since acetate is a more effective pH buffer at pH 5 than pH 6, acetate can be used in smaller amounts in pH 5 solutions than pH 6. In some embodiments, the buffer is at a concentration of about 10 mM ± 5 mM. In particular embodiments, the pH of the composition is from about pH 3 to about pH 7.5, from about pH 3.5 to about pH 7, from about pH 3.5 to about pH 6.5, from about pH 4 to about pH 6, or from about pH 4 to about pH 5, Or about pH 5.0 ± 1.0.

  In other embodiments, the compositions contain isotonicity adjusting agents that make the solution or suspension isotonic for injection and more compatible. Non-limiting examples of isotonic agents include NaCl, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol. In certain embodiments, the isotonic agent is NaCl. In certain embodiments, the NaCl is at a concentration of about 160 ± 20 mM, about 140 mM ± 20 mM, about 120 ± 20 mM, about 100 mM ± 20 mM, about 80 mM ± 20 mM, or about 60 mM ± 20 mM.

  In various embodiments, the composition includes a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In certain embodiments, the preservative is about 0.4% ± 0.2%, about 1% ± 0.5%, about 1.5% ± 0.5%, or about 2.0% ± 0.5. % Concentration.

  In various embodiments, the composition contains an anti-adsorbent (eg, reduces adsorption of CNP variants to glass or plastic). Anti-adsorbents include, but are not limited to, benzyl alcohol, Polysorbate 20, and Polysorbate 80. In certain embodiments, the anti-adsorbent is from about 0.001% to about 0.5%, from about 0.01% to about 0.5%, from about 0.1% to about 1%, from about 0.5% A concentration of about 1%, about 0.5% to about 1.5%, about 0.5% to about 2%, or about 1% to about 2%.

  In various embodiments, the composition includes a stabilizer. Non-limiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and its salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or a salt thereof, the stabilizer is at a concentration of about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer is at a concentration of about 0.01% to about 0.5%, or about 0.01% to about 0.2%. In yet another embodiment, when the stabilizer is glycerin, the stabilizer is at a concentration of about 5% to about 100% (net).

  In various embodiments, the composition contains an antioxidant. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP variant is from about 0.1: 1 to about 15: 1, from about 1: 1 to about 15: 1, from about 0.5: 1 to about 10 :. 1, about 1: 1 to about 10: 1, or about 3: 1 to about 10: 1.

Pharmaceutically acceptable salts may be used in the composition, including but not limited to salts of inorganic acids (eg, hydrochloride, hydrobromide, phosphate, sulfate), salts of organic acids ( For example, acetate, propionate, malonate, benzoate, mesylate, tosylate) and amine (eg, isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine) salts. A thorough discussion of pharmaceutically acceptable ^{salts, Remington's Pharmaceutical Sciences, 18 th} Edition, Mack Publishing Company found in (Easton, Pennsylvania (1990)) .

  The pharmaceutical compositions may be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage form of the composition may be tailored to the desired mode of administration of the composition. For oral administration, the composition may take the form of, for example, a tablet or capsule (including soft gel capsules), for example, an aqueous or non-aqueous solution, suspension, or syrup. Tablets and capsules for oral administration include one or more commonly used excipients, diluents, and carriers such as mannitol, lactose, glucose, sucrose, starch, corn starch, sodium saccharin, talc, cellulose, magnesium carbonate And lubricants (eg, magnesium stearate, sodium stearyl fumarate). If desired, flavoring, coloring, and / or sweetening agents may be added to the solid and liquid formulations. Other optional ingredients for oral formulations include, but are not limited to, preservatives, suspending agents, and thickening agents. The oral formulation may have an enteric coating that protects the CNP variant from the acidic environment of the stomach. Methods for preparing solid and liquid dosage forms are known or will be apparent to those skilled in the art (see, eg, Remington's Pharmaceutical Sciences, referenced above).

  Formulations for parenteral administration may be prepared, for example, as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in liquid medium prior to injection, or as emulsions. For example, injectable sterile solutions and suspensions may be prepared according to techniques known in the art using suitable diluents, carriers, solvents (eg, buffered aqueous solutions, Ringer solutions, saline), dispersing agents, wetting agents, It can be blended using an emulsifier, a suspending agent and the like. Sterile fixed oils, fatty acid esters, polyols, and / or other inert ingredients may also be used. As a further example, formulations for parenteral administration include aqueous sterile injectable solutions, which make antioxidants, buffers, bacteriostatic agents, and formulations isotonic with the blood of the intended recipient. Solutes; and aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents.

  Exemplary CNP formulations are described in US Pat. No. 8,198,242 and US Pat. No. 8,598,121. The use of CNP formulations with a pH in the range of about 4 to about 6 is contemplated.

^１グリセリンは、ＣＮＰ変異体の、水による加水分解、脱アミド、異性化、または切断を最小にし、または防止するのに用いられる。凍結乾燥剤について、４〜６％または６〜２０％のマンニトールまたはスクロースが、ＮａＣｌに置換されてよい。 In various embodiments, the CNP variant may be formulated in a pharmaceutical carrier for administration to a subject suffering from osteoarthritis or symptoms of osteoarthritis. In some embodiments, a CNP variant liquid formulation is formulated according to any combination of ingredients, the amount or concentration of which is described below:

^One glycerin is used to minimize or prevent water hydrolysis, deamidation, isomerization, or cleavage of CNP variants. For the lyophilizer, 4-6% or 6-20% mannitol or sucrose may be replaced with NaCl.

  The composition comprising the CNP variant may be a lyophilizing agent. In certain embodiments, the lyophilizer includes a buffer and a bulk agent, and optionally an antioxidant. Exemplary buffers include, but are not limited to, acetate buffer and citrate buffer. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, mannitol is in an amount of about 3% to about 10%, about 4% to about 8%, or about 4% to about 6%. In certain embodiments, sucrose is in an amount of about 6% to about 20%, about 6% to about 15%, or about 8% to about 12%.

^１グリセリンは、ＣＮＰ変異体の、水による加水分解、脱アミド、異性化、または切断を最小にし、または防止するのに用いられる。 In various embodiments, a CNP variant lyophilizate is prepared from a formulation formulated according to the components described below, and any combination of their amounts or concentrations:

^One glycerin is used to minimize or prevent water hydrolysis, deamidation, isomerization, or cleavage of CNP variants.

  In various embodiments, the formulation comprising the CNP variant has a pH of about 3-7, about 3-6, about 3.5-6.5, about 4-6, about 4-5, or about 4.5- 5.5. In some embodiments, for pH 4-5.5, a suitable buffer is acetic acid / acetate (eg, sodium acetate), and for pH 5.5-6, a suitable buffer is citric acid / citrate. Citric acid / citrate (eg sodium citrate) is also a suitable buffering agent in the pH 3-6 or pH 4-6 range. In certain embodiments, the buffering agent has a concentration in the formulation of about 2-50 mM, about 2-40 mM, about 2-30 mM, about 5-30 mM, about 2-20 mM, about 5-20 mM, or about 5-15 mM. is there.

  To minimize or avoid deamidation of the CNP variant, the variant may be formulated in pharmaceutically acceptable organic cosolvents such as glycerin, ethanol, and propylene glycol. Since deamidation occurs by hydrolysis, the organic co-solvent replaces water to minimize contact of the CNP variant with water. The concentration of the one or more organic solvents in the organic aqueous solvent system can be, for example, about 10% to about 99%, or about 100% if water is not used.

  Also, water may be removed from the formulation by lyophilization to minimize or avoid deamidation of the CNP variant. In some embodiments, the lyophilizer comprises the following ingredients: buffer: sodium acetate and acetic acid, or sodium citrate and citric acid; isotonic / bulking agent: mannitol (eg, 3-10%, 2-8% Or 4-6%); sucrose (e.g., 6-20%, 5-15%, or 8-12%); antioxidants: containing any combination of methionine and / or ascorbic acid, each antioxidant The molar ratio of agent to CNP variant is from about 0.1: 1 to about 1: 1, from about 0.5: 1 to about 5: 1, from about 1: 1 to about 15: 1, from about 1: 1. From about 10: 1, or from about 3: 1 to about 10: 1.

  Deamidation also removes the CNP composition (e.g., liquid formulation or lyophilizer) at lower temperatures, e.g. It can be minimized or avoided by storing at 0 ° C, -60 ° C, -70 ° C, -80 ° C, -90 ° C, or -100 ° C.

  In order to minimize or avoid oxidation of oxidative residues (eg methionine) in CNP variants, the variants may be formulated with one or more antioxidants. Exemplary antioxidants include, but are not limited to, methionine, ascorbic acid, and thioglycerol. Also, for example, oxidation of methionine residues is minimized by purging oxygen from liquid media (if liquid formulation) with nitrogen or argon and / or purging oxygen from container or packaging with nitrogen or argon. Can be prevented or prevented.

  In some embodiments, Polysorbate 20, Polysorbate 80, or benzyl alcohol, or a combination thereof, is a CNP formulation to minimize or prevent adsorption (eg, adsorption of CNP variants to plastic or glass). Added to. In certain embodiments, each anti-adsorbent is about 0.001% to about 0.5%, about 0.01% to about 0.5%, about 0.1% to about 1%, about 0.5% From about 1%, from about 0.5% to about 1.5%, from about 0.5% to about 2%, or from about 1% to about 2%. Exemplary ranges of anti-adsorbents in the formulation include, but are not limited to, about 0.001% to about 0.5% Polysorbate 20, about 0.001% to about 0.5% Polysorbate 80, and / or From about 0.5% to about 1.5% benzyl alcohol.

  In certain embodiments, the liquid CNP formulation comprises: or the lyophilized CNP formulation is prepared from a formulation comprising: (1) having a buffer at a concentration of about 30 mM ± 5 or 10 mM and having a pH of Acetic acid / acetate (eg, sodium acetate) buffer that is about 4 ± 0.5 or 1, and (2) benzyl alcohol (eg, as a preservative and / or anti-adsorbent) at a concentration of about 1% ± 0.5% ), And optionally (3) sucrose at a concentration of about 10% ± 5%.

  The present disclosure also provides kits containing, for example, bottles, vials, ampoules, tubes, cartridges, and / or syringes containing liquid (eg, injectable sterile) or solid (eg, lyophilized) formulations. The kit also administers a solid (eg lyophilized) formulation, including but not limited to reconstitution of the lyophilizate in a syringe for injection or to dilute the concentrate to a lower concentration Pharmaceutically acceptable vehicles or carriers (eg, solvents, solutions, and / or buffers) for reconstitution into solutions or suspensions (eg, by injection) can also be included. In addition, extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules or tablets containing, for example, a CNP-containing composition. The kit may also comprise a dispensing device, such as an aerosol or injection dispensing device, a pen injector, an autoinjector, a needleless injector, a syringe, and / or a needle.

  As a non-limiting example, the kit may comprise a syringe having a single chamber or a dual chamber. For single chamber syringes, the single chamber is a liquid CNP formulation ready for injection, a solid (eg, lyophilized) CNP formulation, or a relatively small amount that can be reconstituted into an injectable solution or suspension. Liquid formulations of CNP variants in a suitable solvent system (eg glycerin) may be included. For dual chamber syringes, one chamber may contain a pharmaceutically acceptable vehicle or carrier (eg, solvent system, solution, or buffer) and the other chamber is the first chamber for injection. A CNP variant solid (eg, lyophilized) CNP formulation, or a liquid in a relatively small amount of a suitable solvent system (eg, glycerin) that can be reconstituted into a solution or suspension using a vehicle or carrier of Formulations may be included.

  As a further example, the kit may comprise one or more pen injector devices or autoinjector devices, and a dual chamber cartridge. One chamber of the cartridge may contain a pharmaceutically acceptable vehicle or carrier (eg, solvent system, solution, or buffer), and the other chamber may be the first chamber vehicle or Contains a CNP variant solid (eg, lyophilized) CNP formulation, or a liquid formulation in a relatively small amount of a suitable solvent system (eg, glycerin), which can be reconstituted into a solution or suspension using a carrier You can do it. The cartridge can contain an amount of CNP variant sufficient for dosing over a desired period of time (eg, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen injector or autoinjector can be adjusted to dispense the desired amount of CNP formulation from the cartridge.

  Also, a pharmaceutical composition comprising a CNP variant is intended to maintain a relatively constant level of dosage over a desired period of time, eg, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. It may be formulated as a sustained release system, a controlled release system, or a sustained release system. Sustained release formulations, controlled release formulations, and sustained release formulations are known in the art, for example, biodegradable polymer systems {eg, hydrophilic polymers [eg, polylactides, polyglycolides, poly ( Lactide-glycolide)], and may take the form of, for example, microparticles, microspheres, or liposomes.

Dosage and Frequency of Dosing As used herein, the term “therapeutically effective amount” of an active agent (eg, a CNP variant) refers to an amount that provides a therapeutic benefit to a patient. The amount can vary from individual to individual and can be determined by several factors including the overall health of the patient. A therapeutically effective amount of a CNP variant can be readily ascertained by one skilled in the art using published materials and procedures. For example, the amount of CNP variant used for treatment should provide an acceptable rate of reversal of cartilage degeneration or increase in cartilage growth.

  The frequency of dosing for a particular individual can vary depending on various factors, including the disorder to be treated and the individual's condition and response to therapy. In certain embodiments, a pharmaceutical composition containing a CNP variant is administered to a subject about once per day, once every two days, once every three days, once per week, twice per week. Administered three times per week, once every two weeks, or monthly.

A CNP variant intended for use herein is administered to a patient at a dose that is therapeutically effective to treat, ameliorate, or prevent osteoarthritis and other symptoms with osteoarthritis-related symptoms. May be. The safety and therapeutic efficacy of CNP variants can be determined by standard pharmacological procedures in cell cultures or experimental animals, for example, LD ₅₀ (dose that kills 50% of the population) and ED ₅₀ (50% of the population). To determine a therapeutically effective dose). The dose ratio between action and therapeutic effect of toxicity is the therapeutic index and it can be expressed as the ratio LD 50 _/ ED _50. Active agents that exhibit large therapeutic indices are usually preferred.

Data obtained from cell culture assays and animal studies can be used to define the range of dosages used in humans. The dosage is usually toxicity little or negligible, falls within a range of circulating concentrations that include the ED _50. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. A therapeutically effective dose can be determined from cell culture assays and animal studies.

  In certain embodiments, the CNP variants described herein are administered at a dose ranging from about 5 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP variant is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, Administer at doses of 450, 500, 750, 1000, 1250, 1500, 1750, or 2000 nmol / kg, or other doses deemed appropriate by the treating physician. In other embodiments, the CNP variant is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190. , 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μg / kg, or about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / Kg dose or at other doses deemed appropriate by the treating physician. The doses of CNP variants described herein include, but are not limited to, daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, every month, etc. May be administered according to dosing frequency / dosing frequency.

  The frequency of CNP variant dosing / administration for a particular subject may vary depending on a variety of factors including the disorder to be treated and the subject's condition and response to therapy. The CNP variant may be administered in a single dose or multiple doses per dose. In certain embodiments, the CNP variant is a single dose or multiple doses daily, every other day, every third day, twice per week, three times per week, weekly, every other week, every third week, every month, Every 6 weeks, every 2 months, every 3 months, or as deemed appropriate by the treating physician.

  In some embodiments, the CNP variant is administered in anticipation of a recovery period that follows the period of growth (eg, chondrogenesis). For example, CNP variants may be administered intravenously, subcutaneously, intraarticularly, or by another mode for a period of time, daily or multiple times per week, followed by a period of no treatment, and The cycle is repeated. In some embodiments, the initial period of treatment (eg, administered daily or multiple CNP variants per week) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks. 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In related embodiments, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the CNP variant dosing regimen is 3 days off daily followed by 3 days off; or 1 week, daily or multiple times per week followed by 3 days off or 1 week off; or 2 weeks daily Or multiple times per week followed by 1 or 2 weeks off; or 3 weeks, daily or multiple times per week followed by 1 week, 2 weeks, or 3 weeks off; or 4 weeks, 5 weeks, 6 weeks 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, daily or multiple times per week, followed by 1 week, 2 weeks, 3 weeks, or 4 weeks off.

Modes of administration CNP variants, or pharmaceutical compositions comprising them, can be obtained by various routes, for example, by subcutaneous, intraarticular, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. It may be administered to a subject. In one aspect, the CNP variant is administered once daily, once every other day, by a single subcutaneous, intraarticular, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. It is administered once a day or once a week.

  CNP variants may also be administered by direct injection at or near the site of disease. In addition, CNP variants may be administered by depot implantation at the target site of action (eg, an abnormal or altered joint or cartilage region). In addition, CNP variants can be delivered sublingually (eg sublingual tablets) under the tongue and delivered into the nasal cavity (eg, intranasal or aerosol spray) by inhalation into the lung (eg, inhaler or aerosol spray). ), By delivery into the eye (eg eye drops), or by transdermal delivery (eg by patches to the skin). CNP variants are microspheres, microcapsules, liposomes (uncharged or charged (eg cation)), polymer microparticles (eg polyamide, polylactide, polyglycolide, poly (lactide-glycolide)) Oral administration may be in the form of a microemulsion or the like.

  Further methods of administration are by osmotic pumps (eg, Alzet pumps) or minipumps (eg, Alzet mini osmotic pumps), which allow controlled control of CNP variants or pharmaceutical compositions over a period of time, Continuous and / or sustained release delivery is possible. The osmotic pump or minipump may be implanted subcutaneously or near the target site (eg, long bones of the limb, epiphysis, etc.).

  It will be apparent to those skilled in the art that CNP variants or compositions thereof can be administered by other modes. Determination of the most effective mode of administration of a CNP variant or composition thereof is within the skill of the artisan.

  CNP variants are, for example, oral (including buccal and sublingual), rectal, nasal, topical, lung, vaginal, or parenteral (intramuscular, intraarterial, intrathecal, subcutaneous, intraarticular, and It may be administered as a pharmaceutical formulation suitable for administration (including intravenous) or in a form suitable for administration by inhalation or insufflation. Depending on the intended mode of administration, the pharmaceutical formulation can be in solid, semi-solid or liquid dosage forms such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, emulsions, creams, ointments, lotions, etc. It may be in the form of The formulation may be provided in unit dosage forms suitable for single administration of precise dosages. The formulation comprises an effective amount of the CNP variant, and one or more pharmaceutically acceptable excipients, carriers, and / or diluents, and optionally one or more other biologically active. Contains agents.

Combination Therapy In one aspect, CNP variants may be combined with one or more other active agents useful for treating, ameliorating, or preventing osteoarthritis. Other active agents can enhance the effects of the CNP variant and / or produce other pharmacological effects other than those of the CNP variant. Non-limiting examples of active agents that can be used in combination with the CNP variants described herein include other natriuretic peptides (eg, BNP), and peptidases and proteases (eg, NEP and furin), NPR-C and There are inhibitors (eg, antagonists) of tyrosine kinases (eg, FGFR-3). Examples of NEP inhibitors include, but are not limited to, thiorphan and candoxatril. Co-use of NPR-C inhibitors can also increase the half-life of CNP mutants through inhibition of mutant clearance by NPR-C.

  CNP variants may be administered in combination with anti-inflammatory or analgesic agents such as anti-inflammatory agents, NSAIDs, corticosteroids, and hyaluronic acid.

  In order to achieve an appropriate therapeutic outcome in combination therapy, one of ordinary skill in the art typically applies a CNP composition and other therapeutic agent to a subject to produce the desired therapeutic outcome (eg, cartilage repair, or reduced cartilage degeneration). ) Will be administered in a combination amount effective to provide. This process may include administering the CNP composition and the other therapeutic agent simultaneously. Simultaneous administration can be accomplished by administering a single composition or pharmacological protein formulation that includes both the CNP variant and the other therapeutic agent. In addition, other therapeutic agents may be taken separately at about the same time as the CNP variant pharmacological formulation (eg, tablet, injection, or beverage). CNP variants may also be formulated in foods suitable for consumption, such as brownies, pancakes, or cakes.

  In other options, administration of the CNP variant may precede or follow administration of the other therapeutic agent by an interval ranging from minutes to hours. In embodiments where the other therapeutic agent and the CNP composition are administered separately, one of skill in the art will generally recognize that the CNP variant and the other therapeutic agent are administered within an appropriate time of each other, and the CNP variant and Both other therapeutic agents will ensure that a beneficial effect on the patient can occur synergistically or additively. For example, one of ordinary skill in the art can administer the CNP composition within about 0.5-6 hours (before or after) of the other therapeutic agent. In one aspect, the CNP composition is administered within about 1 hour (before or after) of the other therapeutic agent.

Patient population identification and monitoring Protocols can be established to identify subjects suitable for CNP treatment. For example, a suitable patient can be identified using joint aspiration / synovial fluid analysis, X-ray, and / or MRI techniques. A protocol can also be established to determine whether a given patient responds to CNP treatment. For example, for the treatment of osteoarthritis, growth indicators such as cartilage growth measurements and motility assessment can be measured.

  One CNP signaling marker is cGMP (guanosine 3 ', 5' cyclic monophosphate). After CNP binds to and activates its cognate receptor NPR-B, the level of this intracellular signaling molecule increases. Increased levels of cGMP are known from cell culture extracts after CNP exposure (in vitro), conditioned media from bone explant tests after CNP exposure (ex vivo), and subcutaneously, intravenously, or known in the art It can be measured in plasma (in vivo) within minutes after CNP administration via other routes of administration.

  A cartilage specific analyte (or cartilage related marker) may also be measured to assess CNP effectiveness. For example, cleaved collagen type II fragments are cartilage-specific markers of cartilage metabolism. Collagen type II is a major organic component of cartilage, and collagen type II fragments (cut collagen) are released into the circulatory system after cartilage metabolism and then secreted into the urine. Cartilage metabolism precedes new bone formation.

  Other potential biomarkers for cartilage formation and growth or degeneration include chondroitin sulfate aggrecan (cartilage specific marker for cartilage metabolism), collagen type II propeptide (cartilage specific marker for cartilage formation), proliferating cell nuclear antigen ( PCNA), chondroitin sulfate aggrecan, syndecan-3, and annexin VI. Cartilage-related biomarkers can be measured using commercially available kits, for example, in serum from efficacy / pharmacodynamic in vivo studies and from conditioned media for ex vivo testing.

  In one aspect, the level of at least one cartilage-related biomarker is assayed or measured in a subject that has been administered a CNP variant to monitor the effect of the CNP variant on cartilage formation and growth in vivo. For example, an increase in the level of at least one bone-related biomarker or cartilage-related biomarker indicates that administration of a CNP variant has a positive effect on bone growth, skeletal dysplasia, and CNP activity. It may be useful in the treatment of other bone-related or cartilage-related diseases or disorders associated with decline. Exemplary cartilage-related biomarkers include, but are not limited to, CNP (eg, endogenous levels of CNP), cGMP, collagen type II propeptide and fragments thereof, collagen type II and fragments thereof, collagen type I propeptide And fragments thereof, collagen type I and fragments thereof, proliferating cell nuclear antigen (PCNA), chondroitin sulfate aggrecan, syndecan-3, and annexin VI.

  In various embodiments, a biomarker is measured by obtaining a biological sample from a subject to which a CNP variant will be administered, being administered, or administered. Biomarkers can be measured using techniques known in the art including, but not limited to, Western blots, enzyme-linked immunosorbent assays (ELISAs), and enzyme activity assays. The biological sample can be blood, serum, urine, or other biological fluid.

  Additional aspects and details of the disclosure will be apparent from the following examples. These are intended to be illustrative rather than limiting.

F. Example Example 1
Rat osteoarthritis model Unilateral medial meniscal rupture in 275-300 gram rats results in rapid progressive cartilage degeneration (by chondrocyte and proteoglycan loss, fibrillation, bone growth, and chondrocyte cloning) Characterized). This model is performed by a transection of the medial collateral ligament just below the attachment to the meniscus so that the meniscus is reflected toward the femur when the joint space is opened. Care should be taken not to damage the meniscus at its narrowest point (away from the ossicles) on the tibial surface and the resulting cross-section is a separate, freely movable anterior meniscus Make sure to produce half and half of the posterior meniscus and cut.

  Progressive cartilage degeneration changes occur up to 3-6 weeks after surgery, and tibial cartilage degeneration can be partially severe in the outer 1/3 of the tibia and in the central and medial 1/3 Is less severe. Bone proliferators eventually become very large in size (medial tibia) and gradually increase. The model is progressive, with loss of total cartilage (resulting in dentinized bone) within 12 months in virtually all rats. Lesions are reasonably consistent. When the weight load is returned to the rat immediately after surgery, gait analysis suggests a slight, if any, change in the load force on the operated knee. Zone analysis may reveal the effect of treatment in the middle and medial 1/3, but due to the rapid progression of cartilage degeneration, the protective effect is not necessarily evident in the lateral 1/3 of the tibial cartilage. Significant subchondral and epiphyseal bone changes occur in the lower medial tibia for areas of greatest lesion severity. These are large, ranging from increased basophilization of the calcified cartilage layer, with less damage to the subchondral bone, to apparent disintegration of the articular cartilage into a region of bone resorption at the epiphysis, where the periphery of the bone has hardened. The size and type vary. Therefore, this model provides an opportunity to evaluate not only the cartilage protective effect of the agent, but also the bone preservation activity. The model is of a relatively short duration and the animals have a very consistent response to surgery.

  This rat model was used to determine the effect of CNP mutants on delaying or repairing cartilage damage in osteoarthritis. There, CNP is delivered in response to joint damage or trauma. In this model, a rupture is introduced into the meniscus, causing joint damage that results in cartilage degeneration and osteoarthritis (Janusz et al., Osteoarthritis Cartilage, 10: 785-791, 2002). Male Lewis rats undergo unilateral medial meniscectomy on day 0; administration of control vehicle or CNP variant Gly-CNP-37 begins on day 7; Euthanized on day 29 for physical analysis. Gly-CNP-37 (provided at 0.71 or 2.2 mg / mL in control vehicle) or control vehicle alone, 3 weeks, 3 times per week (M, W, F), intra-articular (IA) injection Administered. Therapeutic efficacy was assessed by gait analysis and histopathological investigation of chondrocyte death / cartilage degeneration in the knee.

Surgery In detail, surgical animals were anesthetized with isoflurane and the right knee and lower leg regions were prepared for surgery. For meniscal rupture, a skin incision was made across the medial side of the knee and the medial collateral ligament was exposed by blunt dissection and then transected. To simulate a complete rupture and closed wound, the entire layer of the inner meniscus was cut. Ninety minutes after surgery, animals were treated with a dose of acetaminophen (100 mg / kg, PO) in 1% CMC (2 mL / kg, 50 mg / ml). Subsequent doses were given 24 hours after surgery (as needed).

Histopathology For the histopathological analysis, the operated joints were collected from euthanized rats. After 4-6 days in 5% formic acid decalcifier, the operated joint is cut into two roughly equal halves on the front and embedded in paraffin. Sections were cut at approximately 160 μm from each operated right knee and colored with toluidine blue. To evaluate the effect of Gly-CNP-37 on the progression of osteoarthritis, the following parameters were measured or scored for each treated joint and compared to a vehicle treated control joint: cartilage degeneration, lesion depth , Lesion width, damage to calcified cartilage layer and subchondral bone, medial tibia subchondral / apical trabecular bone thickening / hardening, bone growth, collagen damage, growth plate thickness As well as medial collateral ligament / synovial repair.

  For some parameters (if noted), the regional differences across the tibial plateau are considered by dividing each section into three zones (1-outside, 2-medium, 3-inside).

  Data were analyzed using Student's t-test or Mann-Whitney U-test (nonparametric). If appropriate, the data may be further analyzed across the entire group using a one-way analysis of variance (1-way ANOVA) or Kruskal-Wallis test (nonparametric) with appropriate multiple comparison post-tests. . Statistical tests may establish certain hypotheses regarding the normality of the data and the uniformity of the variance and may require further analysis if the test violates this hypothesis. Significance for all tests was set at p <0.05.

General cartilage degeneration of the medial tibia and femur General cartilage degeneration was determined for each joint by assessment of chondrocyte death / loss, proteoglycan (PG) loss, and collagen loss or fibrillation, Scored as shown in Table 1.

(Table 1) Histopathological scoring criteria for cartilage degeneration

  In addition to representing the data for each zone, a total of three zones for cartilage degeneration was calculated. Rats treated with Gly-CNP-37 at low dose (35.5 μg / dose) or high dose (110 μg / dose) showed a significant reduction in cartilage degeneration in the tibia compared to untreated control rats. (FIG. 1).

Depth and width of the medial tibia lesion The depth of any type of lesion (lesion depth) and the depth to the tide mark (total depth) are the largest lesions in each of the three zones of the tibial plateau Measured by eyepiece micrometer in the severity area. From these measurements, the depth ratio was calculated for each joint by dividing the lesion depth by the total depth. The total depth to the tide mark can be an average measurement of cartilage thickness in each of the three zones for comparison of anabolics when measured at the midpoint of the zone. FIG. 2 shows that the depth ratio was improved in the Gly-CNP-37 treated group compared to the vehicle treated control. Cartilage width as measured by eyepiece micrometer, affected by any degeneration (cell loss, proteoglycan loss, or collagen damage) was also improved in the CNP treated group (FIG. 3). This measurement extends from the origin of the bone growth body due to adjacent cartilage degeneration (outside 1/3) across the surface to the point where the tangential layer and underlying cartilage appear histologically normal.

  Significant cartilage degeneration is defined as the loss of chondrocytes and proteoglycans extending over 50% of cartilage thickness as measured with an eyepiece micrometer. Typically, in this model, collagen damage is mild (defined as a depth of up to 25%) or more. However, the loss of chondrocytes and proteoglycans is significant, extending to at least 50% or more of the cartilage depth, indicating a region where permanent structural changes have occurred. The width of the operated joint, showing significant cartilage degeneration, was measured in Gly-CNP-37 treated animals and vehicle treated controls to assess the ability of CNP variants to reduce significant degeneration. As shown in FIG. 4, vehicle-treated controls showed little reduction in degeneration width, while Gly-CNP-37 treated animals showed greater than 50% improvement in significant tibial cartilage degeneration.

Damage to Calcified Cartilage Layer and Subchondral Bone Damage to the calcified cartilage layer and subchondral bone was scored using the criteria in Table 2.

Table 2: Histopathological scoring criteria for cartilage layer and subchondral bone damage

  Treatment with higher doses (110 μg / dose) with Gly-CNP-37 caused calcified cartilage and subchondral bone damage from a score of approximately 2.4 for vehicle treated animals, Gly-CNP-37 treated animals. To 1.3. Also, treatment with lower doses (35.5 μg / dose) with Gly-CNP-37 reduced calcified cartilage and subchondral bone damage, if not higher doses.

Tibia thickening, bone proliferator thickness, and total joint score Thickness / hardening of medial subtibial / epiphyseal trabecular bone based on criteria shown in Table 3, lateral tibia and / or normal left Scores were made using a comparison with the medial tibia.

(Table 3) Histopathological scoring of bone thickening

  The results did not show the significance set by this study, but treatment with both doses with Gly-CNP-37 resulted in medial subtibia subchondral / apical trabecular bone compared to vehicle treated controls. Showed an observable reduction in thickening / hardening.

  The thickness of the bone growth body (tide mark to the farthest point extending towards the synovium) was measured with an eyepiece micrometer. A score was assigned to the largest bone growth in each section (typically found on the tibia) according to the criteria in Table 4.

Table 4: Bone proliferator thickness scoring

  Treatment with high dose (110 μg / dose) with Gly-CNP-37 reduced tibial proliferator measurements by approximately 24% (p <0.05) and reduced tibial proliferator score by approximately 34% ( p <0.05). Bone growth is the growth of bone that occurs in OA joints. Bone proliferator score data demonstrates that CNP variants can reduce bone growth, ie bone growth. This observation is surprising and unexpected given the known role of CNP and CNP variants that promote bone growth.

  The “total joint score” is calculated by summing each of the three zone scores for cartilage degeneration of the tibia and femur and the bone proliferator score (total joint score with femur) and for the three of the tibial cartilage degeneration only Each zone score was calculated for each joint by summing the bone proliferator score (total joint score without femur). As shown in FIG. 5, Gly-CNP-37 at 110 μg / dose significantly reduced the total joint score without femur. Also, treatment with Gly-CNP-37 at the lower 35.5 μg / dose showed an improvement in the total joint score without the femur, if not so, compared to the higher dose. When including a score for the femur (FIG. 6), the results show that Gly-CNP-37 reduces the total joint score, but not to a significant level, at both doses.

Collagen damage, growth plate thickness, and ligament / synovial repair Collagen damage across the medial tibial plateau (two half of the most severely affected sections) was measured using an eyepiece micrometer and the following parameters: Assessed by measuring the following total width: arbitrary damage (fibrillation ranging from surface to full layer loss); severe damage (total loss of collagen to tide mark or nearly total loss, thickness>90%); significant damage (extends through 61-90% of cartilage thickness); moderate damage (extends through 31-60% of cartilage thickness); mild damage (11 of cartilage thickness) Extending through ~ 30%); and minor damage (extremely affects only the top 10% of the surface).

  Treatment with both lower and higher doses with Gly-CNP-37 resulted in fewer areas with severe degeneration, marked degeneration, and moderate degeneration compared to untreated controls. It was.

  The thickness of the growth plate is measured in all knees at the medial side and lateral side (2x) at the approximate midpoint of medial and lateral epiphyseal cartilage (assuming a non-tangential area of the section). Measurement value / joint) was measured using an eyepiece micrometer. Administration of Gly-CNP-37 at a dose of 110 μg significantly reduced the thickness of the inner growth plate. Gly-CNP-37 at a dose of 35.5 μg showed a decrease in inner thickness, but not a significant decrease by statistical analysis (FIG. 7). It was also confirmed by stratified analysis. There, the difference between the two was determined by subtracting the outer thickness from the inner thickness. FIG. 8 shows that using this analysis, Gly-CNP-37 at higher doses significantly reduced growth plate thickness, while lower doses were not as high as higher doses, It shows that the thickness of the growth plate was reduced.

  Inner collateral ligament / synovial repair was also analyzed by measuring the thickness of the inner synovial / collateral ligament repair in the non-tangential region of the section using an eyepiece micrometer. These measurements did not show any difference between treatment groups.

Gait analysis Gait analysis was performed by applying ink to the ventral surface of the foot and recording the weight load (footprint) during movement across the paper. After inking the rat's hind paw, the rat was placed on paper so that it could walk the full length. Gait was scored visually using the criteria in Table 5 (the description refers to the affected / affected leg).

(Table 5) Scoring criteria for gait analysis

  Gly-CNP-37 treated animals showed improved motor performance and gait from approximately day 15 to day 28 of the study at either high or low dose (Figure 9). The area under the curve (AUC) of the walking score was also calculated. FIG. 10 shows that Gly-CNP-37 at the lower dose reduced the gait score much more than the higher CNP dose, and the gait score for both treatment populations compared to the untreated control. It was lower.

  The results shown in Example 1 show that treatment of osteoarthritis with a CNP variant such as Gly-CNP-37 immediately after trauma is beneficial for the symptoms of osteoarthritis, including cartilage degeneration and motility, in affected subjects. It has shown that it has an effect.

Example 2
Additional rat studies were performed using the rat model and experimental methodology of Example 1 to investigate the use of a second CNP variant, Pro-Gly-CNP-37, in the treatment of osteoarthritis, and This was compared with Gly-CNP-37.

(Table 6) Test design and test group

  As summarized in Table 6, groups of rats were treated with vehicle, Pro-Gly-CNP-37, or Gly-CNP-37. The route of administration was intra-articular (IA) or subcutaneous (SC). The dosing schedule was intra-articular administration for 3 weeks (TIW × 3W), 3 times per week (M, W, and F), or subcutaneous administration for approximately 3 weeks (QD × 3W), once daily. Rats experienced surgery on study day 0; groups 1-5 experienced surgery approximately one week prior to groups 6-7 (ie, the study was performed with a staggered shift). While IA injections were made as fixed doses, SC injections were made at a given dose per weight based on the most recently recorded body weight. All rats were weighed prior to dosing. For 300 grams rats, the weekly IA dosing regimens for low, medium and high levels were approximately equal to 15, 50 and 150 μg / kg / day, respectively. Rats were euthanized 25 days after the last dose and joints were collected for histopathological analysis and scoring.

Plasma Pharmacokinetics and Pharmacodynamics The pharmacokinetic (PK) and pharmacodynamic (PD) properties of CNP variants were evaluated. For PK / PD analysis, timed bleeds were performed on the following 3 rats / group / time points: 15, 30, 60 and 120 minutes after dosing for the IA group and for the SC group 5, 15, 30, 60, and 120 minutes after dosing. After blood collection, lavage fluid (synovial lavage fluid: SLF, approximately 50 μL / knee) was collected from the left knee (normal) and right knee (operated) of each rat.

Histopathology and Immunohistochemistry Knees were preserved for histopathology and immunohistochemistry (IHC) analysis. The primary endpoint was histopathological investigation of chondrocyte death and cartilage degeneration in the knee, including IHC for proliferating cell nuclear antigen (PCNA). Various histopathological features were measured and scored as described above in Example 1. In particular, see Tables 1-5 for scoring criteria.

  The total width of the tibial cartilage affected by any degeneration (cell loss, proteoglycan loss, or collagen damage) was measured by an eyepiece micrometer. As shown in FIG. 11, Pro-Gly-CNP-37 given in IA at medium and high doses significantly reduced the total cartilage degeneration width by approximately 26% and 32%, respectively. Therefore, the 35.5 and 105 μg doses with IA reached statistical significance for tibial cartilage degeneration width.

  The effect of the CNP variant on significant cartilage degeneration, defined by chondrocyte and proteoglycan loss extending through more than 50% of cartilage thickness, was measured at the tibial plateau by an eyepiece micrometer. A dose-responsive decrease in significant cartilage degeneration was observed for Pro-Gly-CNP-37 delivered by the intra-articular route (FIG. 12). A 32% reduction in width was seen with the 35.5 μg IA dose and a 36% reduction with the 105 μg IA dose. A statistically significant decrease was observed for 150 μg / kg Pro-Gly-CNP-37 given subcutaneously daily for 3 weeks (42% reduction in width).

  The thickness of the bone growth body (tide mark to the farthest point extending towards the synovium) was measured with an eyepiece micrometer. A dose response trend in the medial tibia bone growth score was observed for Pro-Gly-CNP-37 administered via the IA route (FIG. 13), with a 15% decrease seen at 35.5 μg IA. And a 14% reduction was seen at 105 μg IA. A 26% statistically significant decrease (p <0.05 by t test) was also observed for Pro-Gly-CNP-37 administered subcutaneously. The decrease in bone proliferator score demonstrates that CNP variants can reduce abnormal growth of bone (bone proliferator) in OA joints. Again, this observation is surprising and unexpected as CNP and CNP variants have been shown to promote long bone growth.

  The thickness of the growth plate was measured for each group. The thickness of the inner and outer growth plates was significantly greater in animals IA treated with both 35.5 μg IA and 105 μg IA of Pro-Gly-CNP-37 (FIG. 14). Furthermore, the thickness of the inner and outer growth plates was greater in animals treated SC with Pro-Gly-CNP-37 versus vehicle.

  The total joint score (without femur) was calculated by summing each of the three zone scores for cartilage degeneration of the tibia and the bone proliferator score. As seen in FIG. 15, a significant decrease in joint score severity was observed for IA administration of 35.5 and 105 μg Pro-Gly-CNP-37, and also for 105 μg Gly-CNP-37. . A significant decrease in total joint score (minus femur) was also observed for Pro-Gly-CNP-37 given at SC.

  FIG. 16 shows the synovitis score. Synovitis (mainly mononuclear cell infiltration concentrated inside) was scored using the criteria in Table 7.

(Table 7) Synovitis scoring

  No significant increase in synovitis scoring severity was observed across all dose groups. Endotoxin was below the limit of quantification (<0.5 EU / mL) in all formulations except Gly-CNP-37 (0.78 EU / mL).

  FIG. 17 shows the average change in body weight (grams) from day -4 to day 25 for various groups. A significant decrease in weight gain was observed for Pro-Gly-CNP-37 given at both 35.5 and 105 μg / dose with IA and 150 μg / kg / day with SC.

  FIG. 18 shows the gait analysis score for the group. The gait score was significantly reduced (48%) for Pro-Gly-CNP-37 delivered subcutaneously compared to day 8 vehicle.

FIG. 19 and Table 8 show plasma pharmacokinetics for animals treated with Pro-Gly-CNP-37 (Group 2, Group 3, and Group 4 (IA administration) and Group 7 (SC administration)). Individual plasma samples were analyzed for Pro-Gly-SNP-37 levels and mean concentration versus time profiles were analyzed with Phoenix WinNonlin software using a non-compartmental model of extravascular administration. In FIG. 19, the data for each group was plotted and labeled with the corresponding number of groups (on each line). Pro-Gly-CNP-37 appeared rapidly in the systemic circulatory system after both IA and SC injections. Exposure for IA injections (Group 2, Group 3, and Group 4) were generally proportional to dose with no significant changes in t _1/2 and CL. Exposure was comparable to the lowest IA dose investigated, but once daily SC injection (Group 7) was as effective as the two highest doses of IA injection given 3 times / week Seemed to bring about a reaction.

Table 8: Pharmacokinetics of CNP variants

  The data show that intra-articular treatment with Pro-Gly-CNP-37 (35.5 and 105 μg) or Gly-CNP-37 (105 μg) resulted in histopathological lesions of osteoarthritis caused by rupture of the medial meniscus in rats. , Which has had a significant and beneficial effect.

  Daily subcutaneous treatment with 150 μg / kg / day Pro-Gly-CNP-37 resulted in efficacy that was comparable or better than intra-articular treatment. Overall, this study provides evidence of a cartilage protective effect.

Example 3
A late stage osteoarthritis model was used to assess the effectiveness of Pro-Gly-CNP-37 in previously progressing osteoarthritis (ie, primary osteoarthritis). Osteoarthritis was induced in the hind legs of rats by separating the meniscus as described above in Example 1 and Example 2. However, in contrast to Example 1 and Example 2, lesion pathology was progressed 3 weeks prior to IA treatment or SC treatment with Pro-Gly-CNP-37, resulting in existing OA (ie primary osteoarthritis). ) Was simulated. The overall test design is outlined in Table 9. Specifically, male Lewis rats underwent surgery to rupture the unilateral medial meniscus on study day 0; administration of control vehicle or Pro-Gly-CNP-37 began on study day 21; and The animals were euthanized at the scheduled time after the last dose on study day 22 (groups 1-4) or study day 40 (groups 5-11). The primary endpoint evaluated included a histopathological study of chondrocyte death / cartilage degeneration in the knee.

Table 9: Late stage osteoarthritis study design and group

  All animals survived until the end of the scheduled study. At the end of the study, rats were euthanized, knees were collected, fixed, sectioned for histopathological analysis, and scored as in Tables 1-5 and Table 7. A summary of the test results is shown in Table 10.

  Pro-Gly-CNP-37 (35.5 or 105 μg) -treated IA, treatment with TIWx3W, and Pro-Gly-CNP-37 (150 μg / kg) -treated SC, QDx3W induced late-stage meniscus rupture In osteoarthritis, it showed a slight to significant beneficial effect as judged by assessment of knee histopathology. The results of IA treatment with Pro-Gly-CNP-37 were dose-responsive.

  The lesion severity was slightly increased in the SC control compared to the IA control, except for reduced synovitis in SC treated rats, but the arthritic parameters were generally comparable across the vehicle control group. there were. Body weight gain was significantly increased in the SC vehicle control 40 days after surgery compared to the IA control.

ＣＤ＝軟骨変性、ＩＡ＝関節内、ＳＣ＝皮下、ＴＩＷｘ３Ｗ＝３週間、１週あたり３回、ＱＤｘ３Ｗ＝約３週（２０日）間、毎日１回。
（ＳＥ）＝括弧内に示した標準誤差。
*ｐ＜０．０５ ＡＮＯＶＡ／Ｋ−Ｗ検定（Dunnett/Dunnの事後） 対 各ＩＡビヒクルまたはＳＣビヒクル（４０日目）対照。
†ｐ＜０．０５ Studentのt検定／Ｍ−Ｗ検定 対 ＩＡビヒクル（４０日目）。
‡ｐ＜０．０５ Studentのt検定／Ｍ−Ｗ検定 対 ＳＣビヒクル（４０日目）。 Table 10: Summary of clinical and histopathology data

CD = cartilage degeneration, IA = intra-articular, SC = subcutaneous, TIWx3W = 3 weeks, 3 times per week, QDx3W = once every day for about 3 weeks (20 days).
(SE) = standard error shown in parentheses.
* p <0.05 ANOVA / K-W test (post Dunnett / Dunn) vs. each IA vehicle or SC vehicle (day 40) control.
† p <0.05 Student t test / MW test vs. IA vehicle (day 40).
‡ p <0.05 Student t-test / MW test vs. SC vehicle (day 40).

  Rats treated with IA with 35.5 μg Pro-Gly-CNP-37 had a significant (31%) decrease in the zone-2 tibial cartilage degeneration score compared to the IA vehicle control. The total tibial cartilage degeneration score and significant tibial cartilage degeneration width were reduced by 20% and 30%, respectively, but not significantly. Zone-3 medial tibia cartilage degeneration score and zone-1 femur cartilage degeneration score, when present, were significantly increased, although lesions were generally minor or mild.

  Rats treated with IA with 105 μg Pro-Gly-CNP-37 significantly reduced body weight gain compared to IA vehicle controls. IA treatment with 105 μg Pro-Gly-CNP-37 resulted in cartilage degeneration of zone-2 medial tibia (44% reduction), cartilage degeneration of zone-3 medial tibia (93%) compared to IA vehicle control. ), Total cartilage degeneration of the medial tibia (31%), and total tibial cartilage degeneration width (28%). Significant tibial cartilage degeneration width was reduced (38%) by treatment but not significantly.

  Rats treated with 150 μg / kg Pro-Gly-CNP-37 SC significantly reduced body weight gain compared to SC vehicle control. (SC) treatment with 150 μg / kg Pro-Gly-CNP-37 significantly reduced the total tibial cartilage degeneration width (22% reduction) compared to the SC vehicle control. Significant tibial cartilage degeneration width was reduced, but not significantly, by treatment (16%). The thickness of the inner and outer growth plates increased significantly with treatment and contributed to a significant decrease in the difference between the inner and outer thickness.

  As shown in FIG. 20, Pro-Gly-CNP-37 IA treatment with 105 μg and SC treatment with 150 μg / kg resulted in a decrease in mean cartilage degeneration of the tibia. However, significant tibial cartilage degeneration was reduced by both the IA treatment dose and the highest SC dose (FIG. 21). Furthermore, Pro-Gly-CNP-37-administered IA resulted in a statistically significant reduction in the medial tibia cartilage degeneration score (FIG. 22) and medial tibia cartilage degeneration depth ratio (FIG. 23).

  As previously described in Example 1 and Example 2, total joint scores were calculated for each joint (with and without the femur) by histopathological analysis and scoring. Pro-Gly-CNP-37-administered IA produced a 7% (35.5 μg) and 19% (105 μg) reduction in femoral joint score and 11% (35.5 μg) when calculated without the femur. ) And 21% (105 μg) reduction (FIG. 24 (with femur) and FIG. 25 (without femur)). Pro-Gly-CNP-37 treated SC had a more modest decrease of 7% (with femur) and 10% (without femur) at the highest dose level (150 μg / kg) (FIG. 24 and FIG. 25).

  As seen in FIG. 26, Pro-Gly-CNP-37 treatment had a mild effect on bone proliferator score at all doses and routes of administration. Similar to the early stage osteoarthritis test described in Example 2, Pro-Gly-CNP-37 treatment resulted in a decrease in body weight changes in treated animals (FIG. 27).

  The present invention may be embodied in other specific forms without departing from the true scope of the invention. Any reference to “the present invention” is intended to refer to exemplary embodiments of the invention, and should be construed to refer to all embodiments of the invention, unless the context requires otherwise. Not. The described embodiments are to be considered in all respects only as illustrative and not restrictive; numerous variations and modifications will become apparent to those skilled in the art.

Claims (31)

  A method of treating osteoarthritis in a subject comprising administering a composition comprising a CNP variant to a subject in need thereof.   A method of increasing cartilage growth in a subject comprising administering a composition comprising a CNP variant to a subject in need thereof.   A method of delaying cartilage degeneration in a subject comprising administering to a subject in need thereof a composition comprising a CNP variant.   A method of improving symptoms of osteoarthritis in a subject suffering from osteoarthritis, comprising the step of administering to a subject in need thereof a composition comprising a CNP variant.   A method for improving exercise technique (or exercise capacity) in a patient with osteoarthritis, comprising the step of administering a composition comprising a CNP variant to a subject in need thereof.   A method of inhibiting motor degeneration in a patient with osteoarthritis, comprising the step of administering a composition comprising a CNP variant to a subject in need thereof.   A method of expanding range of motion in a diseased joint of a subject suffering from osteoarthritis, comprising the step of administering a composition comprising a CNP variant to a subject in need thereof.   A method of reducing stiffness in a diseased joint of a subject suffering from osteoarthritis, comprising the step of administering a composition comprising a CNP variant to a subject in need thereof.   A method of alleviating synovitis in a subject comprising administering a composition comprising a CNP variant to a subject in need thereof.   A method of preventing the onset of osteoarthritis in a subject comprising administering to a subject in need thereof a composition comprising a CNP variant.   A method of reducing bone growth body growth in a subject comprising administering a composition comprising a CNP variant to a subject in need thereof. The CNP variant is

The method according to any one of the preceding claims, wherein the method is selected from the group consisting of: The CNP variant is

The method of claim 12, wherein the method is selected from the group consisting of:   The method of claim 1, wherein the osteoarthritis is primary osteoarthritis.   The method of claim 1, wherein the osteoarthritis is secondary osteoarthritis.   15. The method of claim 14, wherein the CNP variant is administered after cartilage degeneration has occurred.   16. The method of claim 15, wherein the CNP variant is administered in response to joint damage or trauma.   18. The method of claim 17, wherein the CNP variant is administered within one month of joint injury or trauma.   The method of any one of the preceding claims, wherein the composition comprising the CNP variant is administered intra-articularly.   The method of any one of the preceding claims, wherein the composition comprising the CNP variant is administered subcutaneously.   The method of any one of the preceding claims, wherein the composition comprising the CNP variant is administered three times a week, twice a week, once a week, or once every two weeks. .   4. The method of claim 2 or 3, wherein the increase in cartilage growth or the delay in cartilage degeneration is observed in the subject's joint.   23. The method of claim 22, wherein the joint is selected from the group consisting of a knee, shoulder, elbow, finger, hand, wrist, crotch, neck, spine, and lower back.   4. The method of claim 2 or 3, wherein cartilage damage or growth is analyzed by chondrocyte death / loss, proteoglycan (PG) loss, or collagen loss or fibrillation.   The method of any one of the preceding claims, wherein the composition comprising the CNP variant further comprises a pharmaceutically acceptable excipient, carrier, or diluent.   26. The method of claim 25, wherein the composition is a lyophilizate prepared from a formulation comprising citrate / citrate buffer or acetic acid / acetate buffer having a pH of about 4 to about 6.   The method according to any one of the preceding claims, further comprising administration of a second agent.   28. The method of claim 27, wherein the second agent is selected from the group consisting of an anti-inflammatory agent, an NSAID, a corticosteroid, and hyaluronic acid.   8. The method of claim 7, wherein the range of motion of the affected joint is determined by measuring hip flexion, hip extension, hip abduction, hip adduction, knee flexion, or knee extension. .   6. The method of any one of the preceding claims, wherein the subject has been identified as having an increased level of at least one cartilage-related biomarker.   The at least one cartilage-related biomarker is CNP, cGMP, collagen type II propeptide and fragments thereof, collagen type II and fragments thereof, osteocalcin, proliferating cell nuclear antigen (PCNA), type I procollagen propeptide (PINP) 31. The method of claim 30, wherein the method is selected from the group consisting of: and fragments thereof, collagen type I and fragments thereof, and chondroitin sulfate aggrecan.

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